Methods and compositions for screening for modulators of IgE synthesis, secretion and switch rearrangement

ABSTRACT

The invention relates to methods and compositions useful in screening for modulators of IgE synthesis, secretion and switch rearrangement.

FIELD OF THE INVENTION

[0001] The invention relates to methods and compositions useful inscreening for modulators of IgE synthesis, secretion and switchrearrangement.

BACKGROUND OF THE INVENTION

[0002] Immunoglobulins must bind to a vast array of foreign moleculesand thus exist in many forms. The sequence of the variable (V) region ofimmunoglobulin molecules varies tremendously, conferring virtuallyunlimited capacity to bind antigens. The constant (C) region comes infive different varieties: α, δ, ε, γ and μ, providing five differentisotypes: IgA, IgD, IgE, IgG and IgM, each of which performs a differentset of functions. B cells initially produce only IgM and IgD, and mustbe activated or induced to produce the other isoforms, such as IgE.

[0003] The course of IgE production starts with the activation of Bcells. Upon activation with an antigen, B cells follow one of twodifferentiation pathways: they may differentiate directly into plasmacells, which are basically antibody-secreting factories, or they maygive rise to germinal centers, specialized structures within lymphoidorgans. In the latter, successive rounds of mutation of the V regiongenes is followed by expression of the gene products on the cellsurface, with selection of the cells on the basis of the affinity of themutated immunoglobulins against the antigen.

[0004] In both pathways of antigen-induced B cell differentiation,isotype switching occurs in which the C region of the immunoglobulinheavy chain changes from the joint expression of IgM and IgD on naive Bcells to expression of one of the downstream isotypes such as IgE. Thisswitching involves the replacement of upstream C regions with adownstream C region that has biologically distinct effector functionswithout changing the structure of the variable portion and, hence, itsspecificity. For IgE switching, a deletional rearrangement of the Igheavy chain gene locus occurs, a rearrangement that joins the switchregion of the μ gene, Sμ, with the corresponding region of the ε gene,Sε. This switching is minimally induced by IL-4 or IL-13, which initatestranscription through the Sε region, resulting in the synthesis ofgerm-line (or “sterile”) ε transcripts; that is, transcripts of theunrearranged C_(ε) heavy genes. This IL-4 induced transcription isinhibited by IFN-γ, IFN-α, and TGF-β. A second signal, normallydelivered by T cells, is required for actual switch recombinationleading to IgE production. The T cell signal may be replaced bymonoclonal antibodies to CD40, Epstein-Barr viral infection, orhydrocortisone.

[0005] Recently, the mechanism of class switch recombination has beenexplained by an accessibility model, wherein the specificity of theswitch gene rearrangement is determined by the modulation of switchregion accessibility; that is, the opening up of the chromatin incertain areas, allowing the required protein/enzyme complexes access tothe genes.

[0006] IgE antibodies are crucial immune mediators of allergicreactions, and have been shown to be responsible for the induction andmaintenance of allergic symptoms. For example, the introduction ofanti-IgE antibodies has been shown to interfere with IgE function, thusworking to alleviate allergic symptoms. See Jardieu, Current Op.Immunol. 7:779-782 (1995), Shields et al., Int. Arch. Allergy. Immunol.107:308-312 (1995). Accordingly, it is an object of the invention toprovide compositions and methods useful in screening for modulators ofIgE production, in particular for modulators of switch rearrangement.

SUMMARY OF THE INVENTION

[0007] In accordance with the objects outlined above, the presentinvention provides methods of screening for bioactive agents capable ofinhibiting an IL-4 inducible ε promoter. The method comprises combininga candidate bioactive agent and a cell comprising a fusion nucleic acid.The fusion nucleic acid comprises an IL-4 inducible ε promoter, and areporter gene. The promoter is then induced with IL-4 (or IL-13), andthe presence or absence of the reporter protein is detected. Generally,the absence of the reporter protein indicates that the agent inhibitsthe IL-4 inducible ε promoter. The fusion nucleic acid may comprise anexogeneous IL-4 inducible ε promoter, or an endogeneous IL-4 inducible εpromoter. Preferred embodiments utilize the use of retroviral vectors tointroduce the candidate bioactive agents.

[0008] In an additional aspect, the present invention provides celllines for screening. Either CA-46 and MC-116 cell lines are included,and further comprise fusion nucleic acids comprising an IL-4 inducible εpromoter, and a reporter gene.

[0009] In a further aspect, the present invention provides methods ofscreening for bioactive agents capable of modulating IgE production. Themethod comprises combining a candidate bioactive agent and a cellcapable of expressing IgE and determining the amount of IgE produced inthe cell. Generally, a change in the amount of IgE as compared to theamount produced in the absence of the candidate agent indicates that theagent modulates IgE production. The cell can further comprise a IgEfusion protein comprises the ε heavy chain, and a fluorescent protein.

[0010] In an additional aspect, the invention provides methods ofscreening for bioactive agents capable of inhibiting a promoter ofinterest. The method comprises combining a candidate bioactive agent anda cell comprising a fusion nucleic acid. The fusion nucleic acidcomprises a promoter of interest and a reporter gene comprising a deathgene that is activated by the introduction of a ligand. The promoter isoptionally induced, and the ligand is introduced to the cell. Thepresence of the cell is then detected, wherein the presence of the cellindicates that the agent inhibits the promoter.

[0011] In a further aspect, the invention provides compositionscomprising a test vector and a reporter vector. The test vectorcomprises a first selection gene, and a fusion gene comprising a firstsequence encoding a transcriptional activation domain, and a secondsequence encoding a test protein. The reporter vector comprises a firstdetectable gene, and all or part of the switch ε sequence, which uponbinding of the transcriptional activation domain due to aprotein-nucleic acid interaction between the test protein and the switchε sequence, will activate transcription of the first detectable gene.Methods utilizing these compositions are also provided; the methodscomprise providing a host cell comprising the composition, andsubjecting the host cell to conditions under which the fusion gene isexpressed to produce a fusion protein. A protein-nucleic acidinteraction between the fusion protein and the switch ε sequence is thendetected.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIGS. 1A and 1B depict the germline ε locus and sequence. FIG. 1Adepicts the sequence of the human IL-4 inducible ε promoter. FIG. 1Bdepicts the organization of the germline ε locus.

[0013]FIGS. 2A and 2B depict the regions (2A) and sequences (2B and 2C)of the switch ε (Sε) region that are used in methods of screening forproteins that interact with the Sε region, as described below.

[0014]FIG. 3 shows a schematic of the yeast one-hybrid system used toidentify proteins that bind to the Sε region.

[0015]FIG. 4 depicts the IL-4 induction of germline ε mRNA in three IgM⁺B cell lines, CA-46, MC-116 and DND39. The cells were incubated for 48hours in 300 U/ml of hIL-4. RT-PCR ws performed using primiers specificfor the germline ε exon and the 5′-end of the ε CH1 exon (predicted sizeis ˜200 bp).

[0016]FIGS. 5A, 5B, 5C and 5D depict two general approaches to generategermline ε promoter knock-in reporter cell lines. FIG. 5A shows theorganization of this region in vivo. FIGS. 5B and 5C depict two possibleknock in constructs. The IL-4 inducible IgM+ B cell lines aretransfected with one or both of these constructs. Under the influence ofIL-4, GFP and/or BFP positive clones are isolated by FACS. Homologousrecombination can be confirmed by PCR and/or Southern blothybridization. FIG. 5D depicts an alternate construct. In thisembodiment, the IL-4 inducible IgM+ B cell lines are transfected withthe 5D construct and selected with G418. Survivors are sorted for thelack of the 3′ BFP expression (deleted during homologous recombination).RT-PCR is performed to confirm homologous recombination. Those clonesare transfected with cre to remove the neomycin resistance gene.

[0017]FIG. 6 depicts a preferred vector for introducing a peptidelibrary into cell lines containing knock-in reporter genes under thecontrol of the IL-4 inducible ε promoter. CRU5 is a modified LTR;Naviaux, et al., “The pCL Vector System: Rapid Production ofHelper-Free, High-Titer, Recombinant Retroviruses,” Journal of Virology,70(8):5701-5705 (1996); LTR=long terminal repeat; ψ+=packaging signal;localization signal=nuclear, cell membrane, etc.; MCS=multiple cloningsite; IRES=internal ribosome entry site; 2a=self-cleaving peptide. Allthe components are cassetted for flexibility.

[0018]FIG. 7 depicts a general schematic of the generation of theprimary peptide libraries in retroviruses.

[0019]FIGS. 8A and 8B depict constructs useful in generating ε heavychain knock-in cell lines.

[0020]FIG. 8A depicts the wild-type organization. FIG. 8B depicts arepresentative construct to produce a GFP knock-in. S=secretory exon;GFP=green fluorescent protein; BFP=blue fluorescent protein;Neo^(r)=neomycin resistance gene; VDJ=V region exon; CH1, 2, 3,4=constant region domain exons; M1, M2=membrane exons; HSV-TK=HerpesSimplex Virus—thymidine kinase.

[0021]FIGS. 9A and 9B depict constructs useful in the invention. FIG. 9Ashows a reporter construct useful to create an IL-4 inducible ε promoterreporter cell line. CRU5=hCMV pormoter plus R and U5 regions of LTR; BGHpoly A=bovine growth hormone poly-adenylation signal;SIN=self-inactivating LTR. FIG. 9B shows a library construct.

[0022]FIGS. 10A and 10B depict a schematic of the screen for candidateagents of the germline ε promoter. FIG. 10A: the experimental schematic.FIG. 10B depicts the survival construct useful in the screen. Position 1can be a number of different genes, including a FAS chimeric receptoroutlined herein (including extracellular mouse Fas receptor or mouse CD8receptor coupled with the human transmembrane and cytoplasmic Fasreceptor), HSV-TK, p450 2B1 and p21 peptide.

[0023]FIGS. 11A, 11B and 11C depict preferred vectors and theirsequences.

[0024]FIGS. 12A, 12B and 12C depict a construct useful in the presentinvention, comprising the a Fas survival construct (i.e. the use of adeath gene). The sequence is of the inducible ε promoter-chimericFas-IRES-hygromycin-bovine growth hormone poly A tail that is put intothe C12s vector backwards to that no leaky transcription happens throughthe cmv promoter.

[0025]FIGS. 13A, 13B and 13C depict a construct useful in the presentinvention, comprising the a Fas survival construct (i.e. the use of adeath gene). The sequence is of the inducible ε promoter-chimeric Fas(either CD8 or mLyt2)-IRES-hygromycin-bovine growth hormone poly A tailthat is put into the C12s vector backwards to that no leakytranscription happens through the cmv promoter.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides compositions and methods useful inscreening for modulators, particularly inhibitors, of the production ofIgE antibodies. In particular, assay methodologies are provided that areamenable to high-throughput screening strategies, such that largenumbers of potential drugs may be screened rapidly and efficiently.Generally, traditional treatments for IgE suppression are based onregulation of the system after IgE has been made, for example usinganti-IgE antibodies or anti-histamines, to modulate the IgE-mediatedresponse resulting in mast cell degranulation. In some cases, drugs areknown that generally downregulate IgE production or that inhibitswitching but not induction of germline transcripts (see for example Lohet al., J. Allerg. Clin. Immunol. 97(5):1141 (1996)).

[0027] In contrast, the present invention provides several relatedtechniques that may be used to screen for upstream modulators of IgEproduction, to prevent the production of IgE and thus reduce oreliminate the allergic response. For example, an early step in the Igswitch is the production of sterile ε transcripts in response to IL-4.It is also appreciated that blockage of the production of membrane boundIgE may induce programmed cell death (PCD). By interfering at this step,highly efficient, rapid and prolonged inhibition of the allergicresponse may occur. In addition, these techniques allow individual cellassessment and thus are useful for high-throughput screening strategies,for example those that utilize fluorescence activated cell sorting(FACS) techniques, and thus allow screening of large numbers ofcompounds for their effects on IgE production.

[0028] In a preferred embodiment, the invention relates to methods thatrely on reporter genes fused to IgE promoters, such as the IL-4inducible ε promoter that starts a cascade that ultimately results inIgE production. Using novel reporter constructs, screening formodulators of this promoter system may be done. Thus the inventionprovides a number of different constructs that allow for screening forantagonists and agonists of these promoters.

[0029] In a preferred embodiment, the invention provides methods ofscreening for bioactive agents capable of modulating, particularlyinhibiting, an IL-4 inducible ε promoter. By “an IL-4 induciblepromoter” herein is meant a nucleic acid promoter that is induced byIL-4, putatively by binding an unknown IL-4 induced DNA binding proteinthat results in induction of the promoter; that is, the introduction ofIL-4 causes the pronounced activation of a particular DNA bindingprotein that then binds to the IL-4 inducible promoter segment andinduces transcription. The sequence of the human IL-4 inducible promoteris shown in FIG. 1, and as will be appreciated by those in the art,derivatives or mutant promoters are included within this definition.Particularly included within the definition of an IL-4 induciblepromoter are fragments or deletions of the sequence shown in FIG. 1. Asis known in the art, the IL-4 inducible promoter is also inducible byIL-13. By “modulating an IL-4 inducible promoter” herein is meant eitheran increase or a decrease (inhibition) of promoter activity, for exampleas measured by the presence or quantification of transcripts or oftranslation products. By “inhibiting an IL-4 inducible promoter” hereinis meant a decrease in promoter activity, with changes of at least about50% being preferred, and at least about 90% being particularlypreferred.

[0030] The methods comprise combining a candidate bioactive agent and acell or a population of cells comprising a fusion nucleic acid. The cellor cells comprise a fusion nucleic acid. In a preferred embodiment, thefusion nucleic acid comprises an IL-4 inducible ε promoter and at leasta first reporter gene. The IL-4 inducible ε promoter is as describedherein, for example SEQ ID NO: 1, or derivatives thereof, and may beeither an endogeneous or exogeneous IL-4 inducible ε promoter, as ismore fully described below.

[0031] By “reporter gene” or “selection gene” herein is meant a genethat by its presence in a cell (i.e. upon expression) can allow the cellto be distinguished from a cell that does not contain the reporter gene.Reporter genes can be classified into several different types, includingdetection genes, survival genes, death genes and cell cycle genes. Itmay be the nucleic acid or the protein expression product that causesthe effect. As is more fully outlined below, additional components, suchas substrates, ligands, etc., may be additionally added to allowselection or sorting on the basis of the reporter gene.

[0032] In a preferred embodiment, the reporter gene encodes a proteinthat can be used as a direct label, i.e. a detection gene, for sortingthe cells, i.e. for cell enrichment by FACS. In this embodiment, theprotein product of the reporter gene itself can serve to distinguishcells that are expressing the reporter gene. In this embodiment,suitable reporter genes include those encoding green fluorescent protein(GFP; Chalfie, et al., “Green Fluorescent Protein as a Marker for GeneExpression,” Science 263(5148):802-805 (Feb. 11, 1994); and EGFP;Clontech—Genbank Accession Number U55762), blue fluorescent protein(BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West,8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H.Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr.Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1.Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif.94303), luciferase (Ichiki, et al.), and β-galactosidase (Nolan, et al.,“Fluorescence-Activated Cell Analysis and Sorting of Viable MammalianCells Based on Beta-D-galactosidase Activity After Transduction ofEscherichia coli LacZ,” Proc Natl Acad Sci USA 85(8):2603-2607 (April1988)).

[0033] Alternatively, the reporter gene encodes a protein that will binda label that can be used as the basis of the cell enrichment (sorting);i.e. the reporter gene serves as an indirect label or detection gene. Inthis embodiment, the reporter gene should encode a cell-surface protein.For example, the reporter gene may be any cell-surface protein notnormally expressed on the surface of the cell, such that secondarybinding agents could serve to distinguish cells that contain thereporter gene from those that do not. Alternatively, albeitnon-preferably, reporters comprising normally expressed cell-surfaceproteins could be used, and differences between cells containing thereporter construct and those without could be determined. Thus,secondary binding agents bind to the reporter protein. These secondarybinding agents are preferably labelled, for example with fluors, and canbe antibodies, haptens, etc. For example, fluorescently labeledantibodies to the reporter gene can be used as the label. Similarly,membrane-tethered streptavidin could serve as a reporter gene, andfluorescently-labeled biotin could be used as the label, i.e. thesecondary binding agent. Alternatively, the secondary binding agentsneed not be labeled as long as the secondary binding agent can be usedto distinguish the cells containing the construct; for example, thesecondary binding agents may be used in a column, and the cells passedthrough, such that the expression of the reporter gene results in thecell being bound to the column, and a lack of the reporter gene (i.e.inhibition), results in the cells not being retained on the column.Other suitable reporter proteins/secondary labels include, but are notlimited to, antigens and antibodies, enzymes and substrates (orinhibitors), etc.

[0034] In a preferred embodiment, the reporter gene is a survival genethat serves to provide a nucleic acid (or encode a protein) withoutwhich the cell cannot survive, such as drug resistant genes. In thisembodiment, the assays may rely on clonal or pooled populations ofcells, since if inhibitors of the promoter are found, the cells willdie, necessitating a clonal population in order to determine thecandidate agent.

[0035] In a preferred embodiment, the reporter gene is a cell cyclegene, that is, a gene that causes alterations in the cell cycle. Forexample, p21 protein its ligand (a collection of three proteins; seeHarper, et al., “The p21 Cdk-Interacting Protein Cip1 Is a PotentInhibitor of G1 Cyclin-Dependent Kinases,” Cell 75:805-816 (Nov. 19,1993)), which does not cause death, but causes cell-cycle arrest, suchthat cells containing inhibited IL-4 inducible promoters grow out muchmore quickly, allowing detection on this basis. As will be appreciatedby those in the art, it is also possible to configure the system suchthat the cells containing the inhibited IL-4 inducible promoters do notgrow out, and thus can be selected on this basis as well.

[0036] In a preferred embodiment, the reporter gene is a death gene thatprovides a nucleic acid that encodes a protein that causes the cells todie. Death genes fall into two basic categories: death genes that encodedeath proteins that require a death ligand to kill the cells, and deathgenes that encode death proteins that kill cells as a result of highexpression within the cell, and do not require the addition of any deathligand. It is preferable that cell death requires a two-step process:the expression of the death gene and induction of the death phenotypewith a signal or ligand, such that the cells may be grown up expressingthe death gene, and then induced to die. A number of death genes/ligandpairs are known, including, but not limited to, the Fas receptor and Fasligand (Bodmer, et al., “Characterization of Fas,” J Biol Chem272(30):18827-18833 (Jul. 25, 1997); muFAS, Gonzalez-Cuadrado, et al.,“Agonistic anti-Fas Antibodies Induce Glomerular Cell Apoptosis in MiceIn Vivo,” Kidney Int 51(6): 1739-1746 (June 1997); Muruva, et al., HumGene Ther, 8(8):955 (May 1997)), (or anti-Fas receptor antibodies); p450and cyclophosphamide (Chen, et al., “Potentiation of CytochromeP450/Cyclophosphamide-Based Cancer Gene Therapy By Coexpression of theP450 Reductase Gene,” Cancer Res 57(21):4830-4837 (Nov. 1, 1997));thymidine kinase and gangcylovir (Stone, R., “Molecular ‘Surgery’ ForBrain Tumors,” 256(5063):1513 (Jun. 12, 1992)), tumor necrosis factor(TNF) receptor and TNF. Alternatively, the death gene need not require aligand, and death results from high expression of the gene; for example,the overexpression of a number of programmed cell death (PCD) proteinsare known to cause cell death, including, but not limited to, caspases,bax, TRADD, FADD, SCK, MEK, etc.

[0037] As will be appreciated by those in the art, the use of the deathgenes in the manner described herein, particularly in two-stepapplications, allows general and high-throughput screening forinhibitors of other promoters, in addition to the IL-4 inducible εpromoters described herein. Thus, the present invention provides fusionnucleic acids comprising a promoter of interest operably linked to adeath gene for use in screening methods. The promoter of interest can beeither a constitutive promoter or an inducible promoter, such as theIL-4 inducible ε promoter. As will be appreciated by those in the art,any number of possible promoters could be used. Suitable promoters ofinterest include, but are not limited to, inducible promoters such asIL-4 ε promoter, promoters that are induced by cytokines or growthfactors such as the interferon responsive factors 1 to 4, NFkB (Fiering,et al., “Single Cell Assay of a Transcription Factor Reveals a Thresholdin Transcription Activated By Signals Emanating From the T-Cell AntigenReceptor,” Genes Dev 4(10):1823-1834 (October 1990)), etc. Wheninducible promoters are used in this embodiment, suitable cell types arethose that can be induced by the appropriate inducer, as will beappreciated by those in the art.

[0038] Preferred embodiments fall into one of three configurations. In apreferred embodiment, the promoter of interest is a constitutivepromoter, and it is hooked to a death gene that requires the presence ofa ligand, such as Fas or TNF. Thus, the cells can be grown up and thepresence of the death gene verified due to the constitutive promoter.This is generally done by hooking the death gene up to a detection genesuch as GFP or BFP, etc., using either an IRES or a protease cleavagesite as is outlined below; thus, the presence of the detection genemeans the death gene is also present. Verification of the presence ofthe death gene is preferred to keep the levels of false positives low;that is, cells that survive the screen should be due to the presence ofan inhibitor of the promoter rather than a lack of the death gene.

[0039] Once the cells have been enriched for those containing the deathgene, the candidate agents can be added (and their presence verified aswell), followed by induction in the presence of IL-4, and finally byaddition of the death ligand. Thus, the cell population is enriched forthose cells that have an agent that inhibits the promoter and thus doesnot produce the death protein, i.e. those that survive.

[0040] Alternatively, a preferred embodiment utilizes fusion nucleicacids comprising promoters of interest that are inducible (such as theIL-4 ε promoter), and hooked to a death gene that requires a deathligand. The presence of the death gene is verified by inducing thepromoter, causing the death gene (and preferably a detection gene) to bemade. The candidate agents and death ligands are then introduced in thepresence of their appropriate inducer, and the population is enrichedfor those cells that survive, i.e. contain an agent that inhibits thepromoter and thus does not produce the death protein.

[0041] When death genes that require ligands are used, i.e. for “twostep” processes, preferred embodiments utilize chimeric death genes,i.e. chimeric death receptor genes. These chimeric death receptorscomprise the extracellular domain of a ligand-activated multimerizingreceptor and the endogeneous cytosolic domain of a death receptor gene,such as Fas or TNF. This is done to avoid endogeneous activation of thedeath gene. The mechanism of Fas-induced cell death involves theintroduction of the Fas ligand, which can bind two monomeric Fasreceptors, causing the multimerization of the receptor, which activatesthe receptor and leads to secondary signalling resulting in caspaseactivation and PCD. However, as will be appreciated by those in the art,it is possible to substitute the extracellular portion of the deathreceptor with the extracellular portion of another ligand-activatedmultimerizing receptor, such that a completely different signalactivates the cell to die. There are a number of known ligand-activateddimerizing receptors, including, but not limited to, the CD8 receptor,erythropoeitin receptor, thrombopoeitin receptor, growth hormonereceptor, Fas receptor, platelet derived growth hormone receptor,epidermal growth factor receptor, leptin receptor, and a variety ofinterleukin receptors (including, but not limited to, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, andIL-17; although the use of the IL-4 and IL-13 receptors are notpreferred, since these can be used to induce the promoter and thus doesnot provide a “two step” death process), low-density lipoproteinreceptor, prolactin receptor, and transferrin receptor.

[0042] In a preferred embodiment, chimeric Fas receptor genes are made.The exact combination will depend on the cell type used and thereceptors normally produced by these cells. For example, when usinghuman cells or cell lines, a non-human extracellular domain and a humancytosolic domain are preferred, to prevent endogeneous induction of thedeath gene. For example, a preferred embodiment utilizes human cells, amurine extracellular Fas receptor domain and a human cytosolic domain,such that the endogeneous human Fas ligand will not activate the murinedomain. Alternatively, human extracellular domains may be used when thecells used do not endogeneously produce the ligand; for example, thehuman EPO extracellular domain may be used when the cells do notendogeneously produce EPO. (Kawaguchi, et al., Cancer Lett., 116(1):53(1997); Takebayashi, et al., Cancer Res., 56(18):4164 (1996); Rudert, etal., Biochem Biophys Res Commun., 204(3):1102 (1194); Rudert, et al.,DNA Cell Biol., 16(2):197 (1997); Takahasi, et al., J Biol Chem.271(29):17555 (1996); Adam, et al., J Biol Chem., 268(26):19882 (1993);Mares, et al., Growth Factors, 6(2):93 (1992); Seedorf, et al., J BiolChem., 266(19):12424 (1991); Heidaran, et al., J Biol Chem.,265(31):18741 (1990); Okuda, et al., J Clin Invest. 100(7):1708 (1997);Allgood, et al., Curr Opin Biotechnol., 8(4):474 (1997); Anders, et al.,J Biol Chem., 271(36):21758 (1996); Krishnan, et al., Oncogene,13(1):125 (1996); Declercq, et al., Cytokine, 7(7):701 (1995); Bazzoni,et al., Proc Natl Acad Sci US., 92(12):5380 (1995); Ohashi, et al., ProcNatl Acad Sci USA, 91(1):158 (1994); Desai, et al., Cell, 73(3):541(1993); and Amara, et al., Proc Natl Acad Sci USA, 94(20):10618 (1997)).

[0043] In addition to the extracellular domain and the cytosolic domain,these receptors have a transmembrane domain. As will be appreciated bythose in the art, for chimeric death receptor genes, the transmembranedomain from any of the receptors can be used, although in general, it ispreferred to use the transmembrane domain associated with the chosencytosolic domain, to preserve the interaction of the transmembranedomain with other endogeneous signalling proteins.

[0044] Thus, preferred embodiments provide fusion nucleic acids thatutilize the IL-4 inducible ε promoter linked to a death gene,particularly a chimeric death receptor gene, that requires a deathligand for cell killing.

[0045] Alternatively, inducible promoters can be linked to “one step”death genes, i.e. death genes that upon a certain threshold expression,will kill a cell without requiring a ligand or secondary signal. In thisembodiment, the inducible promoter is preferably “leaky”, such that somesmall amount of death gene and a required secondary reporter gene suchas a survival gene or a detection gene can be expressed. The cells thatcontain the death gene can then be selected on this basis, to avoidfalse positives. Once the presence of the construct is verified,candidate agents are added (and their presence preferably verified,using a detection or selection gene as well), and the promoter isinduced. The population is then enriched for those cells that containagents that inhibit the promoter, i.e. that will survive.

[0046] In a preferred embodiment, additional reporter genes are used,particularly when inducible death genes are used. In a preferredembodiment, the additional reporter gene is a selection gene. The cellscontaining the death gene and the drug selectable gene are grown; if theappropriate drug is added to the culture, only those cells containingthe resistance gene (and hence the death gene) survive. This ensuresthat the cells are expressing the death gene to decrease “falsepositives”, i.e. cells that do not die because they do not contain thedeath gene.

[0047] In an additional preferred embodiment, the additional reportergene is a labeling gene such as GFP. The use of a detection gene allowscells to be sorted to give a population enriched for those containingthe construct. As outlined above,a preferred embodiment uses “leaky”inducible promoters; that is, the cells are selected such that the IL-4inducible promoter, even in the absence of IL-4 or IL-13, produces someGFP and death gene (for example, the Fas receptor constructs). In thisembodiment, suitably “leaky” promoters are chosen such that some GFP isexpressed (preferably enough to select the cells expressing theconstruct from those that are not), but not enough death gene isproduced to cause death. While preferred embodiments utilize death genesrequiring the addition of a death ligand, it is well known that highlevels of some death genes, even in the absence of death ligand, cancause death. Thus, for example, high levels of Fas receptor expressioncan cause multimerization, and thus activation, even in the absence ofthe Fas ligand.

[0048] In a preferred embodiment, when two reporter genes are used, theyare fused together in such a way as to only require a single promoter,and thus some way of functionally separating the two genes is preferred.This can be done on the RNA level or the protein level. Preferredembodiments utilize either IRES sites (which allows the translation oftwo different genes on a single transcript (Kim, et al., “Constructionof a Bifunctional mRNA in the Mouse By Using the Internal RibosomalEntry Site of the Encephalomycarditis Virus,” Molecular and CellularBiology 12(8):3636-3643 (August 1992) and McBratney, et al., “TheSequence Context of the Initiation Codon in the EncephalomycarditisVirus Leader Modulates Efficiency of Internal Translation Initiation,”Current Opinion in Cell Biology 5:961-965 (1993)), or a proteasecleavage site (which cleaves a protein translation product into twoproteins).

[0049] Preferred protease cleavage sites include, but are not limitedto, the 2a site (Ryan et al., J. Gen. Virol. 72:2727 (1991); Ryan etal., EMBO J. 13:928 (1994); Donnelly et al., J. Gen. Virol. 78:13(1997); Hellen et al., Biochem, 28(26):9881 (1989); and Mattion et al.,J. Virol. 70:8124 (1996), all of which are expressly incorporated byreference), prosequences of retroviral proteases including humanimmunodeficiency virus protease and sequences recognized and cleaved bytrypsin (EP 578472, Takasuga et al., J. Biochem. 112(5)652 (1992))factor X_(a) (Gardella et al., J. Biol. Chem. 265(26):15854 (1990), WO9006370), collagenase (J03280893, Tajima et al., J. Ferment. Bioeng.72(5):362 (1991), WO 9006370), clostripain (EP 578472), subtilisin(including mutant H64A subtilisin, Forsberg et al., J. Protein Chem.10(5):517 (1991), chymosin, yeast KEX2 protease (Bourbonnais et al., J.Bio. Chem. 263(30):15342 (1988), thrombin (Forsberg et al., supra; Abathet al., BioTechniques 10(2): 178 (1991)), Staphylococcus aureus V8protease or similar endoproteinase-Glu-C to cleave after Glu residues(EP 578472, Ishizaki et al., Appl. Microbiol. Biotechnol. 36(4):483(1992)), cleavage by NIa proteainase of tobacco etch virus (Parks etal., Anal. Biochem. 216(2):413 (1994)), endoproteinase-Lys-C (U.S. Pat.No. 4,414,332) and endoproteinase-Asp-N, Neisseria type 2 IgA protease(Pohlner et al., Bio/Technology 10(7):799-804 (1992)), soluble yeastendoproteinase yscF (EP 467839), chymotrypsin (Altman et al., ProteinEng. 4(5):593 (1991)), enteropeptidase (WO 9006370), lysostaphin, apolyglycine specific endoproteinase (EP 316748), and the like. See e.g.Marston, F. A. O. (1986) Biol. Chem. J. 240, 1-12.

[0050] In addition to the promoter of interest, such as an IL-4inducible ε promoter and reporter gene, the fusion nucleic acids maycomprise additional components, including, but not limited to, otherreporter genes, protein cleavage sites, internal ribosome entry (IRES)sites, AP-1 sites, and other components as will be appreciated by thosein the art.

[0051] In a preferred embodiment, foreign constructs comprising the IL-4inducible ε promoter and the reporter gene are made. By “foreign” hereinis meant that the fusion nucleic acids originates outside of the cells.That is, a recombinant nucleic acid is made that contains an exogeneousIL-4 inducible ε promoter and a reporter gene. Thus, in somecircumstances, the cells will contain both exogeneous and endogeneousIL-4 inducible ε promoters. By “recombinant nucleic acid” herein ismeant nucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated nucleic acid, in a linear form, anucleic acid containing components not normally joined, such as an IL-4inducible promoter and a reporter gene, or an expression vector formedin vitro by ligating DNA molecules that are not normally joined, are allconsidered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention. In thisembodiment, any cells that express an IL-4 receptor that transduces theIL-4 signal to the nucleus and alters transcription can be used.Suitable cells include, but are not limited to, human cells and celllines that show IL-4/13 inducible production of germline ε transcripts,including, but not limited to, DND39 (see Watanabe, supra), MC-116,(Kumar, et al., “Human BCGF-12kD Functions as an Autocrine Growth Factorin Transformed B Cells,” Eur Cytokine Netw 1(2):109 (1990)), CA-46(Wang, et al., “UCN-01: A Potent Abrogator of G2 Checkpoint Function inCancer Cells with Dirupted p53,” J Natl Cancer Inst 88:956 (1996)).

[0052] This recombinant nucleic acid may introduced to a cell in avariety of ways, as will be appreciated by those in the art, including,but not limited to, CaPO₄ precipitation, liposome fusion, lipofectin®,electroporation, viral infection, etc. The constructs may preferablystably integrate into the genome of the host cell (for example, withretroviral introduction, outlined below), or may exist eithertransiently or stably in the cytoplasm (i.e. through the use oftraditional plasmids, utilizing standard regulatory sequences, selectionmarkers, etc.).

[0053] In a preferred embodiment, the exogeneous constructs, which maybe in the form of an expression vector, are added as retroviralconstructs, using techniques generally described in PCT US97/01019 andPCT US97/01048, both of which are expressly incorporated by reference,and the examples.

[0054] In a preferred embodiment, the fusion construct comprises anendogeneous IL-4 inducible ε promoter and an exogeneous reporter gene;“endogeneous” in this context means originating within the cell. Thatis, gene “knock-in” constructions are made, whereby an exogeneousreporter gene as outlined herein is added, via homologous recombination,to the genome, such that the reporter gene is under the control of theendogeneous IL-4 inducible ε promoter. This may be desirable to allowfor the exploration and modulation of the full range of endogeneousregulation, i.e. regulatory elements (particularly those flanking thepromoter) other than just the IL-4 inducible ε promoter fragment.Exemplary constructs are shown in FIGS. 5B and 5C, with GFP and BFP,although other reporter genes outlined herein may be used.

[0055] Homologous recombination may proceed in several ways. In oneembodiment, traditional homologous recombination is done, with molecularbiological techniques such as PCR being done to find the correctinsertions. For example, gene “knock-ins” may be done as is known in theart, for example see Westphal et al., Current Biology 7:R530-R533(1997), and references cited therein, all of which are expresslyincorporated by reference. The use of recA mediated systems may also bedone, see PCT US93/03868, hereby expressly incorporated by reference.

[0056] Alternatively, and preferably, the selection of the “knock ins”are done by FACS on the basis of the incorporation of a reporter gene.Thus, in a preferred embodiment, a first homologous recombination eventis done to put a first reporter gene, such as GFP, into at least oneallele of the cell genome. Preferably, this is a cell type that exhibitsIL-4 inducible production of at least germline ε transcripts, so thatthe cells may be tested by IL-4 production for reporter gene expression.Suitable cells include, but are not limited to, human cells and celllines that show IL-4/13 inducible production of germline ε transcripts,including, but not limited to, DND39 (see Watanabe, supra), MC-116,(Kumar, et al., “Human BCGF-12kD Functions as an autocrine Growth Factorin Transformed B Cells,” Eur Cytokine Netw 1(2):109 (1990)), CA-46(Wang, et al., “UCN-01 :A Potent Abrogator of G2 Checkpoint Function inCancer Cells with Dirupted p53,” J Natl Cancer Inst 88:956 (1996)). Asis noted herein, the ability of MC-116 and CA-46 cells to producegermline ε transcripts upon IL-4/13 induction was not known prior to thepresent invention. Thus, preferred embodiments provide MC-116 and/orCA-46 cells comprising recombinant nucleic acid reporter constructs areoutlined herein.

[0057] In a preferred embodiment, once a first endogeneous promoter hasbeen combined with an exogeneous reporter construct, a second homologousrecombination event may be done, preferably using a second reporter genedifferent from the first, such as BFP, to target the other allele of thecell genome, and tested as above.

[0058] Generally, IL-4 induction of the reporter genes will indicate thecorrect placement of the genes, which can be confirmed via sequencingsuch as PCR sequencing or Southern blot hybridization. In addition,preferred embodiments utilize prescreening steps to remove “leaky”cells, i.e. those showing constitutive expression of the reporter gene.

[0059] Thus, in a preferred embodiment, the invention provides celllines that contain fusion nucleic acids comprising IL-4 inducible εpromoter operably connected to at least one reporter gene. Once made,the cell lines comprising these reporter constructs are used to screencandidate bioactive agents for the ability to modulate the production ofIgE, as is outlined below.

[0060] The term “candidate bioactive agent” or “exogeneous compound” asused herein describes any molecule, e.g., protein, oligopeptide, smallorganic molecule, polysaccharide, polynucleotide. Generally a pluralityof assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection.

[0061] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Particularly preferred are peptides.

[0062] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification,amidification to produce structural analogs.

[0063] In a preferred embodiment, the candidate bioactive agents areproteins. By “protein” herein is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

[0064] In a preferred embodiment, the candidate bioactive agents arenaturally occuring proteins or fragments of naturally occuring proteins.Thus, for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eucaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

[0065] In a preferred embodiment, the candidate bioactive agents arepeptides of from about 5 to about 30 amino acids, with from about 5 toabout 20 amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccuring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

[0066] In one embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

[0067] In a preferred embodiment, the candidate bioactive agents arenucleic acids. By “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein means at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805(1984), Letsinger, et al, J. Am. Chem. Soc., 110:4470 (1988); andPauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc., 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl.,31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al.,Nature, 380:207 (1996), all of which are incorporated by reference)).Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionicbackbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141;and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English,30:423 (1991); Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988);Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, etal., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occuring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xathanine hypoxathanine, isocytosine, isoguanine, etc.

[0068] As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occuring nucleic acids, random nucleicacids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eucaryotic genomes may be used as is outlined above forproteins.

[0069] In a preferred embodiment, the candidate bioactive agents areorganic chemical moieties, a wide variety of which are available in theliterature.

[0070] In a preferred embodiment, a library of different candidatebioactive agents are used. Preferably, the library should provide asufficiently structurally diverse population of randomized agents toeffect a probabilistically sufficient range of diversity to allowbinding to a particular target. Accordingly, an interaction libraryshould be large enough so that at least one of its members will have astructure that gives it affinity for the target. Although it isdifficult to gauge the required absolute size of an interaction library,nature provides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. A library of all combinations of a peptide 7 to 20 aminoacids in length, such as generally proposed herein, has the potential tocode for 20⁷ (10⁹) to 20²⁰. Thus, with libraries of 10⁷ to 10⁸ differentmolecules the present methods allow a “working” subset of atheoretically complete interaction library for 7 amino acids, and asubset of shapes for the 20²⁰ library. Thus, in a preferred embodiment,at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ andmost preferably at least 10⁹ different sequences are simultaneouslyanalyzed in the subject methods. Preferred methods maximize library sizeand diversity.

[0071] The candidate bioactive agents are combined or added to a cell orpopulation of cells. Suitable cell types for different embodiments areoutlined above. By “population of cells” herein is meant at least twocells, with at least about 10⁵ being preferred, at least about 10⁶ beingparticularly preferred, and at least about 10⁷, 10⁸ and 10⁹ beingespecially preferred.

[0072] The candidate bioactive agent and the cells are combined. As willbe appreciated by those in the art, this may accomplished in any numberof ways, including adding the candidate agents to the surface of thecells, to the media containing the cells, or to a surface on which thecells are growing or in contact with; adding the agents into the cells,for example by using vectors that will introduce the agents into thecells (i.e. when the agents are nucleic acids or proteins).

[0073] In a preferred embodiment, the candidate bioactive agents areeither nucleic acids or proteins (proteins in this context includesproteins, oligopeptides, and peptides) that are introduced into the hostcells using retroviral vectors, as is generally outlined in PCTUS97/01019 and PCT US97/01048, both of which are expressly incorporatedby reference. Generally, a library of retroviral vectors is made usingretroviral packaging cell lines that are helper-defective and arecapable of producing all the necessary trans proteins, including gag,pol and env, and RNA molecules that have in cis the ψ packaging signal.Briefly, the library is generated in a retrovirus DNA constructbackbone; standard oligonucleotide synthesis is done to generate eitherthe candidate agent or nucleic acid encoding a protein, for example arandom peptide, using techniques well known in the art. After generationof the DNA library, the library is cloned into a first primer. The firstprimer serves as a “cassette”, which is inserted into the retroviralconstruct. The first primer generally contains a number of elements,including for example, the required regulatory sequences (e.g.translation, transcription, promoters, etc), fusion partners,restriction endonuclease (cloning and subcloning) sites, stop codons(preferably in all three frames), regions of complementarity for secondstrand priming (preferably at the end of the stop codon region as minordeletions or insertions may occur in the random region), etc.

[0074] A second primer is then added, which generally consists of someor all of the complementarity region to prime the first primer andoptional necessary sequences for a second unique restriction site forsubcloning. DNA polymerase is added to make double-strandedoligonucleotides. The double-stranded oligonucleotides are cleaved withthe appropriate subcloning restriction endonucleases and subcloned intothe target retroviral vectors, described below.

[0075] Any number of suitable retroviral vectors may be used. Generally,the retroviral vectors may include: selectable marker genes under thecontrol of internal ribosome entry sites (IRES) that greatly facilitatesthe selection of cells expressing peptides at uniformly high levels; andpromoters driving expression of a second gene, placed in sense oranti-sense relative to the 5′ LTR. Suitable selection genes include, butare not limited to, neomycin, blastocidin, bleomycin, puromycin, andhygromycin resistance genes, as well as self-fluorescent markers such asgreen fluorescent protein, enzymatic markers such as lacZ, and surfaceproteins such as CD8, etc.

[0076] Preferred vectors include a vector based on the murine stem cellvirus (MSCV) (see Hawley et al., Gene Therapy 1:136 (1994)) and amodified MFG virus (Rivere et al., Genetics 92:6733 (1995)), and pBABE,outlined in the examples.

[0077] The retroviruses may include inducible and constitutive promotersfor the expression of the candidate agent (to be distinguished from theIL-4 inducible ε promoter). For example, there are situations wherein itis necessary to induce peptide expression only during certain phases ofthe selection process. A large number of both inducible and constitutivepromoters are known.

[0078] In addition, it is possible to configure a retroviral vector toallow inducible expression of retroviral inserts after integration of asingle vector in target cells; importantly, the entire system iscontained within the single retrovirus. Tet-inducible retroviruses havebeen designed incorporating the Self-Inactivating (SIN) feature of 3′LTR enhancer/promoter retroviral deletion mutant (Hoffman et al., PNASUSA 93:5185 (1996)). Expression of this vector in cells is virtuallyundetectable in the presence of tetracycline or other active analogs.However, in the absence of Tet, expression is turned on to maximumwithin 48 hours after induction, with uniform increased expression ofthe whole population of cells that harbor the inducible retrovirus,indicating that expression is regulated uniformly within the infectedcell population. A similar, related system uses a mutated TetDNA-binding domain such that it bound DNA in the presence of Tet, andwas removed in the absence of Tet. Either of these systems is suitable.

[0079] In a preferred embodiment, the candidate bioactive agents arelinked to a fusion partner. By “fusion partner” or “functional group”herein is meant a sequence that is associated with the candidatebioactive agent, that confers upon all members of the library in thatclass a common function or ability. Fusion partners can be heterologous(i.e. not native to the host cell), or synthetic (not native to anycell). Suitable fusion partners include, but are not limited to: a)presentation structures, as defined below, which provide the candidatebioactive agents in a conformationally restricted or stable form; b)targeting sequences, defined below, which allow the localization of thecandidate bioactive agent into a subcellular or extracellularcompartment, particularly a nuclear localization sequence (NLS); c)rescue sequences as defined below, which allow the purification orisolation of either the candidate bioactive agents or the nucleic acidsencoding them; d) stability sequences, which confer stability orprotection from degradation to the candidate bioactive agent or thenucleic acid encoding it, for example resistance to proteolyticdegradation; e) dimerization sequences, to allow for peptidedimerization; f) reporter genes (preferably a labeling gene or asurvival gene); or g) any combination of a), b), c), d), e), or f) aswell as linker sequences as needed.

[0080] In a preferred embodiment, the fusion partner is a presentationstructure. By “presentation structure” or grammatical equivalents hereinis meant a sequence, which, when fused to candidate bioactive agents,causes the candidate agents to assume a conformationally restrictedform. Proteins interact with each other largely through conformationallyconstrained domains. Although small peptides with freely rotating aminoand carboxyl termini can have potent functions as is known in the art,the conversion of such peptide structures into pharmacologic agents isdifficult due to the inability to predict side-chain positions forpeptidomimetic synthesis. Therefore the presentation of peptides inconformationally constrained structures will benefit both the latergeneration of pharmaceuticals and will also likely lead to higheraffinity interactions of the peptide with the target protein. This facthas been recognized in the combinatorial library generation systemsusing biologically generated short peptides in bacterial phage systems.A number of workers have constructed small domain molecules in which onemight present randomized peptide structures.

[0081] While the candidate bioactive agents may be either nucleic acidor peptides, presentation structures are preferably used with peptidecandidate agents. Thus, synthetic presentation structures, i.e.artificial polypeptides, are capable of presenting a randomized peptideas a conformationally-restricted domain. Generally such presentationstructures comprise a first portion joined to the N-terminal end of therandomized peptide, and a second portion joined to the C-terminal end ofthe peptide; that is, the peptide is inserted into the presentationstructure, although variations may be made, as outlined below. Toincrease the functional isolation of the randomized expression product,the presentation structures are selected or designed to have minimalbiologically activity when expressed in the target cell.

[0082] Preferred presentation structures maximize accessibility to thepeptide by presenting it on an exterior loop. Accordingly, suitablepresentation structures include, but are not limited to, minibodystructures, loops on beta-sheet turns and coiled-coil stem structures inwhich residues not critical to structure are randomized, zinc-fingerdomains, cysteine-linked (disulfide) structures, transglutaminase linkedstructures, cyclic peptides, B-loop structures, helical barrels orbundles, leucine zipper motifs, etc.

[0083] In a preferred embodiment, the presentation structure is acoiled-coil structure, allowing the presentation of the randomizedpeptide on an exterior loop. See, for example, Myszka et al., Biochem.33:2362-2373 (1994), hereby incorporated by reference). Using thissystem investigators have isolated peptides capable of high affinityinteraction with the appropriate target. In general, coiled-coilstructures allow for between 6 to 20 randomized positions.

[0084] A preferred coiled-coil presentation structure is as follows:MGCAALESEVSALESEVAS LE SEVAALGRGDMPLAAVKS KL SAVKSKLASVKSKLAACGPP. Theunderlined regions represent a coiled-coil leucine zipper region definedpreviously (see Martin et al., EMBO J. 13(22):5303-5309 (1994),incorporated by reference). The bolded GRGDMP region represents the loopstructure and when appropriately replaced with randomized peptides(i.e.candidate bioactive agents, generally depicted herein as (X)_(n),where X is an amino acid residue and n is an integer of at least 5 or 6)can be of variable length. The replacement of the bolded region isfacilitated by encoding restriction endonuclease sites in the underlinedregions, which allows the direct incorporation of randomizedoligonucleotides at these positions. For example, a preferred embodimentgenerates a XhoI site at the double underlined LE site and a HindIIIsite at the double-underlined KL site.

[0085] In a preferred embodiment, the presentation structure is aminibody structure. A “minibody” is essentially composed of a minimalantibody complementarity region. The minibody presentation structuregenerally provides two randomizing regions that in the folded proteinare presented along a single face of the tertiary structure. See forexample Bianchi et al., J. Mol. Biol. 236(2):649-59 (1994), andreferences cited therein, all of which are incorporated by reference).Investigators have shown this minimal domain is stable in solution andhave used phage selection systems in combinatorial libraries to selectminibodies with peptide regions exhibiting high affinity, Kd=10⁻⁷, forthe pro-inflammatory cytokine IL-6.

[0086] A preferred minibody presentation structure is as follows:MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDT SQSILYLQKKKGPP.The bold, underline regions are the regions which may be randomized. Theitalized phenylalanine must be invariant in the first randomizingregion. The entire peptide is cloned in a three-oligonucleotidevariation of the coiled-coil embodiment, thus allowing two differentrandomizing regions to be incorporated simultaneously. This embodimentutilizes non-palindromic BstXI sites on the termini.

[0087] In a preferred embodiment, the presentation structure is asequence that contains generally two cysteine residues, such that adisulfide bond may be formed, resulting in a conformationallyconstrained sequence. This embodiment is particularly preferred whensecretory targeting sequences are used. As will be appreciated by thosein the art, any number of random sequences, with or without spacer orlinking sequences, may be flanked with cysteine residues. In otherembodiments, effective presentation structures may be generated by therandom regions themselves. For example, the random regions may be“doped” with cysteine residues which, under the appropriate redoxconditions, may result in highly crosslinked structured conformations,similar to a presentation structure. Similarly, the randomizationregions may be controlled to contain a certain number of residues toconfer β-sheet or α-helical structures.

[0088] In a preferred embodiment, the fusion partner is a targetingsequence that targets the candidate bioactive agent to a particularsubcellular location. As will be appreciated by those in the art, thelocalization of proteins within a cell is a simple method for increasingeffective concentration and determining function. The concentration of aprotein can also be simply increased by nature of the localization.Shuttling the proteins into the nucleus confines them to a smaller spacethereby increasing concentration. While other targeting sequences suchas targeting sequences to the Golgi, endoplasmic reticulum, nuclearmembrane, mitochondria, secretory vesicles, lysosome, and cellularmembrane may be used, a preferred embodiment uses targeting sequences tothe nucleus, i.e. a nuclear localization signal (NLS).

[0089] In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val), Kalderon (1984),et al., Cell, 39:499-509; the human retinoic acid receptor-β nuclearlocalization signal (ARRRRP); NFκB p50 (EEVQRKRQKL; Ghosh et al., Cell62:1019 (1990); NFκB p65 (EEKRKRTYE; Nolan et al., Cell 64:961 (1991);and others (see for example Boulikas, J. Cell. Biochem. 55(1):32-58(1994), hereby incorporated by reference) and double basic NLS'sexemplified by that of the Xenopus (African clawed toad) protein,nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln AlaLys Lys Lys Lys Leu Asp), Dingwall, et al., Cell, 30:449-458, 1982 andDingwall, et al., J. Cell Biol., 107:641-849; 1988). Numerouslocalization studies have demonstrated that NLSs incorporated insynthetic peptides or grafted onto reporter proteins not normallytargeted to the cell nucleus cause these peptides and reporter proteinsto be concentrated in the nucleus. See, for example, Dingwall, andLaskey, Ann, Rev. Cell Biol., 2:367-390, 1986; Bonnerot, et al., Proc.Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et al., Proc. Natl.Acad. Sci. USA, 87:458-462, 1990.

[0090] In a preferred embodiment, the fusion partner is a rescuesequence. A rescue sequence is a sequence which may be used to purify orisolate either the candidate agent or the nucleic acid encoding it.Thus, for example, peptide rescue sequences include purificationsequences such as the His₆ tag for use with Ni affinity columns andepitope tags for detection, immunoprecipitation or FACS(fluoroscence-activated cell sorting). Suitable epitope tags include myc(for use with the commercially available 9E10 antibody), the BSPbiotinylation target sequence of the bacterial enzyme BirA, flu tags,lacZ, and GST.

[0091] Alternatively, the rescue sequence may be a uniqueoligonucleotide sequence which serves as a probe target site to allowthe quick and easy isolation of the retroviral construct, via PCR,related techniques, or hybridization.

[0092] In a preferred embodiment, the fusion partner is a stabilitysequence to confer stability to the candidate bioactive agent or thenucleic acid encoding it. Thus, for example, peptides may be stabilizedby the incorporation of glycines after the initiation methionine (MG orMGG0), for protection of the peptide to ubiquitination as perVarshavsky's N-End Rule, thus conferring long half-life in thecytoplasm. Similarly, two prolines at the C-terminus impart peptidesthat are largely resistant to carboxypeptidase action. The presence oftwo glycines prior to the prolines impart both flexibility and preventstructure initiating events in the di-proline to be propagated into thecandidate peptide structure. Thus, preferred stability sequences are asfollows: MG(X)_(n)GGPP, where X is any amino acid and n is an integer ofat least four.

[0093] In one embodiment, the fusion partner is a dimerization sequence.A dimerization sequence allows the non-covalent association of onerandom peptide to another random peptide, with sufficient affinity toremain associated under normal physiological conditions. Thiseffectively allows small libraries of random peptides (for example, 10⁴)to become large libraries if two peptides per cell are generated whichthen dimerize, to form an effective library of 10⁸ (10⁴×10⁴). It alsoallows the formation of longer random peptides, if needed, or morestructurally complex random peptide molecules. The dimers may be homo-or heterodimers.

[0094] Dimerization sequences may be a single sequence thatself-aggregates, or two sequences, each of which is generated in adifferent retroviral construct. That is, nucleic acids encoding both afirst random peptide with dimerization sequence 1, and a second randompeptide with dimerization sequence 2, such that upon introduction into acell and expression of the nucleic acid, dimerization sequence 1associates with dimerization sequence 2 to form a new random peptidestructure.

[0095] Suitable dimerization sequences will encompass a wide variety ofsequences. Any number of protein-protein interaction sites are known. Inaddition, dimerization sequences may also be elucidated using standardmethods such as the yeast two hybrid system, traditional biochemicalaffinity binding studies, or even using the present methods.

[0096] In a preferred embodiment, the fusion partner is a detectiongene, preferably a labeling gene or a survival gene. That is, it isdesirable to know that the candidate bioactive agent is a) present andb) being expressed. Thus, preferred embodiments utilize fusionconstructs utilizing genes that allow the detection of cells thatcontain candidate bioactive agents, as is generally outlined in theExamples, and shown in FIG. 10. Preferred detection genes include, butare not limited to, GFP, BFP, YFP, RFP, luciferase, and β-galactosidase.Preferred embodiments utilize detection genes that are different fromthe reporter genes used to determine whether the IL-4 inducible promoteris inhibited; that is, if a GFP reporter gene is used, preferably anon-GFP detection gene is used. This allows cell enrichment using FACSthat can distinguish between cells containing candidate agents and thosethat do not, as well distinguishing cells containing candidate agentsthat do not inhibit the promoter and cells containing candidate agentsthat do inhibit the promoter.

[0097] In a preferred embodiment, as for the other constructs outlinedherein, when a detection gene fusion partner is used with nucleic acidencoding a peptide candidate agent (which may also include other fusionpartners as described herein), the two nucleic acids are fused togetherin such a way as to only require a single promoter, i.e. using either anIRES site or a protease cleavage site such as 2a. A preferred embodimentis depicted in FIG. 10B.

[0098] The fusion partners may be placed anywhere (i.e. N-terminal,C-terminal, internal) in the structure as the biology and activitypermits.

[0099] In a preferred embodiment, the fusion partner includes a linkeror tethering sequence, as generally described in PCT US 97/01019, thatcan allow the candidate agents to interact with potential targetsunhindered. For example, when the candidate bioactive agent is apeptide, useful linkers include glycine-serine polymers (including, forexample, (GS)_(n), (GSGGS)_(n) and (GGGS)_(n), where n is an integer ofat least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers such as the tether for the shaker potassiumchannel, and a large variety of other flexible linkers, as will beappreciated by those in the art. Glycine-serine polymers are preferredsince both of these amino acids are relatively unstructured, andtherefore may be able to serve as a neutral tether between components.Secondly, serine is hydrophilic and therefore able to solubilize whatcould be a globular glycine chain. Third, similar chains have been shownto be effective in joining subunits of recombinant proteins such assingle chain antibodies.

[0100] In addition, the fusion partners, including presentationstructures, may be modified, randomized, and/or matured to alter thepresentation orientation of the randomized expression product. Forexample, determinants at the base of the loop may be modified toslightly modify the internal loop peptide tertiary structure, whichmaintaining the randomized amino acid sequence.

[0101] In a preferred embodiment, combinations of fusion partners areused. Thus, for example, any number of combinations of presentationstructures, targeting sequences, rescue sequences, and stabilitysequences may be used, with or without linker sequences.

[0102] Thus, candidate agents can include these components, and may thenbe used to generate a library of fragments, each containing a differentrandom nucleotide sequence that may encode a different peptide. Theligation products are then transformed into bacteria, such as E. coli,and DNA is prepared from the resulting library, as is generally outlinedin Kitamura, PNAS USA 92:9146-9150 (1995), hereby expressly incorporatedby reference.

[0103] Delivery of the library DNA into a retroviral packaging systemresults in conversion to infectious virus. Suitable retroviral packagingsystem cell lines include, but are not limited to, the Bing and BOSC23cell lines described in WO 94/19478; Soneoka et al., Nucleic Acid Res.23(4):628 (1995); Finer et al., Blood 83:43 (1994); Pheonix packaginglines such as PhiNX-eco and PhiNX-ampho, described below; 292T+gag-poland retrovirus envelope; PA317; and cell lines outlined in Markowitz etal., Virology 167:400 (1988), Markowitz et al., J. Virol. 62:1120(1988), Li et al., PNAS USA 93:11658 (1996), Kinsella et al., Human GeneTherapy 7:1405 (1996), all of which are incorporated by reference.Preferred systems include PhiNX-eco and PhiNX-ampho or similar celllines, disclosed in PCT US97/01019.

[0104] In general, the candidate agents are added to the cells underreaction conditions that favor agent-target interactions. Generally,this will be physiological conditions. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high through put screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away.

[0105] A variety of other reagents may be included in the assays. Theseinclude reagents like salts, neutral proteins, e.g. albumin, detergents,etc which may be used to facilitate optimal protein-protein bindingand/or reduce non-specific or background interactions. Also reagentsthat otherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may beused. The mixture of components may be added in any order that providesfor the requisite binding.

[0106] Once the candidate agents have been introduced or combined withthe cells containing the fusion constructs, the IL-4 inducible εpromoter is induced. Alternatively, the promoter is induced prior to theaddition of the candidate bioactive agents, or simultaneously. This isgenerally done as is known in the art, and involves the addition of IL-4or IL-13 to the cells at a concentration of not less than 5 units/mlwith 200 units/ml being most preferred. Addition of IL-4 or IL-13 isusually 24-48 hours after the bioactive agents are added.

[0107] The presence or absence of the reporter gene is then detected.This may be done in a number of ways, as will be appreciated by those inthe art, and will depend in part on the reporter gene. For example,cells expressing a label reporter gene, such as GFP, can bedistinguished from those not expressing the gene, and preferably sorted(enriched by FACS) on this basis. Similarly, cells expressing the deathgene will die, leaving only cells that have inhibited promotion of theexpression of the gene, etc. In general, the cells that express thereporter gene (i.e. non-inhibited IL-4 inducible ε promoter) andseparated from those that do not (i.e. the IL-4 inducible ε promoter wasinhibited). This may be done using FACS, lysis selection usingcomplements, cell cloning, scanning by a Fluorimager, growth under drugresistance, enhanced growth, etc.

[0108] In a preferred embodiment, for example when the reporter gene isa death gene, sorting of cells containing bioactive agents that inhibitthe IL-4 inducible ε promoter (and thus do not turn on the death gene)from those cells that contain candidate agents that do not inhibit thepromoter is simple: only those surviving cells contain such an agent.

[0109] In a preferred embodiment, the presence or absence of thereporter gene is determined using a fluorescent-activated cell sorter(FACS). In general, the expression of the reporter gene comprising alabel (or allowing the use of a label) is optimized to allow forefficient enrichment by FACS. Thus, for example, in general, 10 to 1000fluores per sorting event are needed; i.e. per cell, with from about 100to 1000 being preferred, and from 500 to 1000 being especiallypreferred. This can be accomplished by amplifying the signal perreporter gene, i.e. have each second label comprise multiple fluores, orby having a high density of reporter genes per cell; or a combination ofboth.

[0110] In a preferred embodiment, the cells are sorted at very highspeeds, for example greater than about 5,000 sorting events per sec,with greater than about 10,000 sorting events per sec being preferred,and greater than about 25,000 sorting events per second beingparticularly preferred, with speeds of greater than about 50,000 to100,000 being especially preferred. The use of multiple laser pathsallows sort accuracy of 1 in 10⁶ with better than 70% accuracy.

[0111] The sorting results in a population of cells containing thereporter protein (i.e. the promoter was not inhibited) and at least onepopulation of cells without the reporter protein (i.e. the promoter wasinhibited). The absence of the reporter protein is indicative that atleast one candidate bioactive agent is a bioactive agent that inhibitsthe IL-4 inducible ε promoter.

[0112] In addition to screening methods utilizing the reporterconstructs described above, the invention also provides methods forscreening candidate agents for the ability to modulate IgE production.By “modulating IgE production” herein is meant either an increase or adecrease in IgE production, as quantified by the amount of IgE proteinmade. In this embodiment, cells that have already switched to the εheavy chain region can no longer be blocked at the earlier phase of IgEproduction. This is especially important for memory B cells thatmaintain their capacity to secrete IgE and are long lived. Thus, in thisembodiment, candidate agents are screened to identify compounds that canblock IgE at the level of ε heavy chain transcription, translation,assembly and trafficking, to prevent the terminal stages of IgEproduction. In this embodiment, a candidate bioactive agent is combinedwith a cell capable of expressing IgE, preferably surface IgE. Preferredcells include, but are not limited to, cells that produce surface IgEsuch as the U266 cell line (Lagging, et al., “Distribution of PlasmaCell Markers and Intracellular IgE in Cell Line U266,” ImmunologyLetters 49:71 (1996)).

[0113] The candidate agent and the cells are combined, as outlinedabove, and the cells screened for alterations in the amount of IgEproduced, as compared to the amount produced in the absence of thecandidate bioactive agent. This may be done using standard IgE labelingtechniques, including, but not limited to, the use of anti-IgEantibodies, that may be either directly or indirectly labeled, forexample through the use of fluorescent anti-IgE antibodies orfluorescent secondary antibodies, and through the use of IgE fusionproteins, as outlined below.

[0114] In a preferred embodiment, the amount of IgE produced isdetermined through the use of IgE fusion proteins; that is, the IgE isproduced as a fusion protein comprising the IgE protein, specifically atleast the ε heavy chain, and a detectable protein such as is generallyoutlined above for label reporter genes. In a preferred embodiment, gene“knock in” cell lines are produced, as outlined above and shown in theFigures. In this embodiment, a first label gene, such as the gene forgreen fluorescent protein (GFP), is fused to the secretory exon of IgEto label secretory IgE heavy chains green. In a preferred embodiment, asecond label gene, such as the gene for blue fluorescent protein (BFP),is attached to the M2 exon to label membrane IgE heavy chains blue. Thisis preferred as it allows discrimination between mRNA processing andtranslation of secretory versus membrane ε-heavy chain transcripts.Suitable label genes for this embodiment include, but are not limitedto, GFP, BFP, YFP and RFP.

[0115] Accordingly, the present invention provides cell lines thatproduce fusion proteins comprising IgE (either secreted or membranebound) fused to a label protein, preferably a fluorescent protein.

[0116] In yet another preferred embodiment, the invention providesmethods of identifying proteins that bind to all or part of the switch εregion (FIG. 2B). The general idea is to use a “one hybrid” system toidentify proteins that bind to all or part of the switch ε region. Tothis end, the present invention provides compositions comprising a testvector and a reporter vector, and cells containing these vectors. Thesecells may be yeast, such as YM4271 or any yeast cell lines that reporterconstructs can be inserted into.

[0117] By “vector” or “episome” herein is meant a replicon used for thetransformation of host cells. The vectors may be either self-replicatingextrachromosomal vectors (“plasmids”) or vectors which integrate into ahost genome. A preferred embodiment utilizes retroviral vectors, as ismore fully described below.

[0118] Suitable vectors will depend on the host cells used. For use ofthe system in yeast, suitable vectors are known in the art and include,but are not limited to, pHisi-1 and pLacZi (Clonetech Cat #K1603-1) (Li,et al., “Isolation of ORC6, A Component of the Yeast Origin ofRecognition Complex By a One-Hybrid System,” Science 262:1870-1873(1993); Liu, et al. “Identifying DNA-Binding Sites and AnalyzingDNA-Binding Domains Using a Yeast Selection System,” In: Methods: ACompanion to Methods in Enzymology 5:125-137 (1993), Luo, et al.,“Cloning and Analysis of DNA-Binding Proteins By Yeast One-Hybrid andOne-Two-Hybrid Systems,” Biotechniques 20:564-568 (1996), and Strubin,et al., “OBF-1, A Novel B Cell-Specific Coactivator That StimulatesImmunoglobin Promoter Activity Through Association with Octamer-BindingProteins,” Cell 80:497-506 (1995)). Yeast expression systems are wellknown in the art, and include expression vectors for Saccharomycescerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha,Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P.pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferredpromoter sequences for expression in yeast include the inducible GAL1,10promoter, the promoters from alcohol dehydrogenase, enolase,glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

[0119] For non-retroviral mammalian cell embodiments, suitable vectorsare derived from any number of known vectors, including, but not limitedto, pCEP4 (Invitrogen), pCI-NEO (Promega), and pBI-EGFP (Clontech).Basically, any mammalian expression vectors with strong promoters suchas CMV can be used to construct test vectors.

[0120] In a preferred embodiment, one or more retroviral vectors areused. Currently, the most efficient gene transfer methodologies harnessthe capacity of engineered viruses, such as retroviruses, to bypassnatural cellular barriers to exogenous nucleic acid uptake. The use ofrecombinant retroviruses was pioneered by Richard Mulligan and DavidBaltimore with the Psi-2 lines and analogous retrovirus packagingsystems, based on NIH 3T3 cells (see Mann et al., Cell 33:153-159(1993), hereby incorporated by reference). Such helper-defectivepackaging lines are capable of producing all the necessary transproteins -gag, pol, and env- that are required for packaging,processing, reverse transcription, and integration of recombinantgenomes. Those RNA molecules that have in cis the ψ packaging signal arepackaged into maturing virions.

[0121] Retroviruses are preferred for a number of reasons. First, theirderivation is easy. Second, unlike Adenovirus-mediated gene delivery,expression from retroviruses is long-term (adenoviruses do notintegrate). Adeno-associated viruses have limited space for genes andregulatory units and there is some controversy as to their ability tointegrate. Retroviruses therefore offer the best current compromise interms of long-term expression, genomic flexibility, and stableintegration, among other features. The main advantage of retroviruses isthat their integration into the host genome allows for their stabletransmission through cell division. This ensures that in cell typeswhich undergo multiple independent maturation steps, such ashematopoietic cell progression, the retrovirus construct will remainresident and continue to express. In addition, transfection efficienciescan be extremely high, thus obviating the need for selection genes insome cases.

[0122] A particularly well suited retroviral transfection system isdescribed in Mann et al., supra: Pear et al., PNAS USA 90(18):8392-6(1993); Kitamura et al., PNAS USA 92:9146-9150 (1995); Kinsella et al.,Human Gene Therapy 7:1405-1413; Hofmann et al., PNAS USA 93:5185-5190;Choate et al., Human Gene Therapy 7:2247 (1996); WO 94/19478; PCTUS97/01019, and references cited therein, all of which are incorporatedby reference.

[0123] Any number of suitable retroviral vectors may be used. Preferredretroviral vectors include a vector based on the murine stem cell virus(MSCV) (see Hawley et al., Gene Therapy 1:136 (1994)) and a modified MFGvirus (Rivere et al., Genetics 92:6733 (1995)), and pBABE (see PCTUS97/01019, incorporated by reference). Particularly preferred vectorsare shown in FIG. 11.

[0124] As for the other vectors, the retroviral vectors may includeinducible and constitutive promoters. Constitutive promoters arepreferred for the bait and test vectors, and include, but are notlimited to, CMV, SV40, Srα, RSV, and TK. Similarly, the reporter vectorpromoter is associated with at least one copy of an operator, asoutlined herein.

[0125] In addition, it is possible to configure a retroviral vector toallow expression of bait genes or test genes after integration of a baitor test vector in target cells. For example, Tet-inducible retrovirusescan be used to express bait or test genes (Hoffman et al., PNAS USA93:5185 (1996)). Expression of this vector in cells is virtuallyundetectable in the presence of tetracycline or other active analogs.However, in the absence of Tet, expression is turned on to maximumwithin 48 hours after induction, with uniform increased expression ofthe whole population of cells that harbor the inducible retrovirus,indicating that expression is regulated uniformly within the infectedcell population. A similar, related system uses a mutated TetDNA-binding domain such that it bound DNA in the presence of Tet, andwas removed in the absence of Tet. Either of these systems is suitable.

[0126] Generally, these expression vectors include transcriptional andtranslational regulatory nucleic acid operably linked to nucleic acidswhich are to be expressed. “Operably linked” in this context means thatthe transcriptional and translational regulatory nucleic acid ispositioned relative to any coding sequences in such a manner thattranscription is initiated. Generally, this will mean that the promoterand transcriptional initiation or start sequences are positioned 5′ tothe coding region. The transcriptional and translational regulatorynucleic acid will generally be appropriate to the host cell used, aswill be appreciated by those in the art. Numerous types of appropriateexpression vectors, and suitable regulatory sequences, are known in theart for a variety of host cells.

[0127] In general, the transcriptional and translational regulatorysequences may include, but are not limited to, promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, and enhancer or activatorsequences. In a preferred embodiment, the regulatory sequences include apromoter and transcriptional start and stop sequences.

[0128] Promoter sequences encode either constitutive or induciblepromoters. The promoters may be either naturally occurring promoters,hybrid or synthetic promoters. Hybrid promoters, which combine elementsof more than one promoter, are also known in the art, and are useful inthe present invention.

[0129] In general, the vectors of the present invention utilize twodifferent types of promoters. In a preferred embodiment, the promoterson the bait and test vectors are constitutive, and drive the expressionof the fusion proteins and selection genes, if applicable, at a highlevel. However, it is possible to utilize inducible promoters for thefusion constructs and selection genes, if necessary.

[0130] The test vector comprises a selection gene. Selection genes allowthe selection of transformed host cells containing the vector, andparticularly in the case of mammalian cells, ensures the stability ofthe vector, since cells which do not contain the vector will generallydie. Selection genes are well known in the art and will vary with thehost cell used. Suitable selection genes include, but are not limitedto, neomycin, blastocidin, bleomycin, puromycin, hygromycin, and otherdrug resistance genes, as well as genes required for growth on certainmedia, including, but not limited to, His and Lev or His and Trp. Insome cases, for example when using retroviral vectors, the requirementfor selection genes is lessened due to the high transformationefficiencies which can be achieved. Accordingly, selection genes neednot be used in retroviral constructs, although they can be. In addition,when retroviral vectors are used, the test vectors may also containdetectable genes as are described herein rather than selection genes; itmay be desirable to verify that the vector is present in the cell, butnot require selective pressure for maintenance.

[0131] In addition to the selection gene, the test vector comprises afusion gene comprising a first sequence encoding a transcriptionalactivation domain, and a second sequence encoding a test protein. By“fusion gene” or “fusion construct” herein is meant nucleic acid thatcomprises at least two functionally distinct sequences; i.e. generallysequences from two different genes. As will be appreciated by those inthe art, in some embodiments the sequences described herein may be DNA,for example when extrachromosomal plasmids are the vectors, or RNA, forexample when retroviral vectors are used. Generally, the sequences aredirectly linked together without any linking sequences, although in someembodiments linkers such as restriction endonuclease cloning sites orlinkers encoding flexible amino acids such as glycine and serine linkerssuch as are known in the art are used. In a preferred embodiment, thefirst fusion gene comprises a first sequence encoding a transcriptionalactivation domain. By “transcriptional activator domain” herein is meanta proteinaceous domain which is able to activate transcription.

[0132] Suitable transcription activator domains include, but are notlimited to, transcriptional activator domains from GAL4 (amino acids1-147; see Fields et al., Nature 340:245 (1989), and Gill et al., PNASUSA 87:2127 (1990)), GCN4 (from S. cerevisiae, Hope et al., Cell 46:885(1986)), ARD1 (from S. cerevisiae, Thukral et al., Mol. Cell. Biol.9:2360 (1989)), the human estrogen receptor (Kumar et al., Cell 51:941(1987)), VP16 (Triezenberg et al., Genes Dev. 2(6):718-729 (1988)), andB42 (Gyuris et al, Cell 1993), and NF-kB p65, derivatives thereof whichare functionally similar.

[0133] The fusion nucleic acid also includes a test nucleic acid,encoding a test protein. By “test protein” herein is meant a candidateprotein which is to be tested for interaction with a bait protein.Protein in this context means proteins, oligopeptides, and peptides,i.e. at least two amino acids attached. In a preferred embodiment, thetest protein sequence is one of a library of test protein sequences;that is, a library of test proteins is tested for binding to one or morebait proteins. The test protein sequences can be derived from genomicDNA, cDNA or can be random sequences. Alternatively, specific classes oftest proteins may be tested. The library of test proteins or sequencesencoding test proteins are incorporated into a library of test vectors,each or most containing a different test protein sequence.

[0134] In a preferred embodiment, the test protein sequences are derivedfrom genomic DNA sequences. Generally, as will be appreciated by thosein the art, genomic digests are cloned into test vectors. The genomiclibrary may be a complete library, or it may be fractionated or enrichedas will be appreciated by those in the art.

[0135] In a preferred embodiment, the test protein sequences are derivedfrom cDNA libraries. A cDNA library from any number of different cellsmay be used, and cloned into test vectors. As above, the cDNA librarymay be a complete library, or it may be fractionated or enriched in anumber of ways.

[0136] In a preferred embodiment, the test protein sequences are randomsequences. Generally, these will be generated from chemicallysynthesized oligonucleotides. Generally, random test proteins range insize from about 2 amino acids to about 100 amino acids, with from about10 to about 50 amino acids being preferred. Fully random or “biased”random proteins may be used; that is, some positions within the sequenceare either held constant or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., forzinc fingers, SH-2 domains, stem loop structures, or to purines, or toreduce the chance of creation of a stop codon, etc.

[0137] The compositions of the invention also include reporter vectors.Generally, the test and reporter vectors are distinct, although as willbe appreciated by those in the art, one or two independent vectors maybe used. The reporter vectors comprise a first detectable or reportergene and all or part of the switch ε sequence, which functions as anoperator site. That is, upon binding of a test protein to the switch εsequence (i.e. a protein-nucleic acid interaction), the transcriptionalactivator domain of the fusion protein will activate transcription andcause expression of the selectable or detectable gene(s). Thus, in thisembodiment, the test protein functions essentially as a candidate agent.

[0138] In a preferred embodiment, the compositions are introduced intohost cells to screen for protein-nucleic acid interactions. By“introduced into” or grammatical equivalents herein is meant that thenucleic acids enter the cells in a manner suitable for subsequentexpression of the nucleic acid. The method of introduction is largelydictated by the targeted cell type and the composition of the vector.Exemplary methods include CaPO₄ precipitation, liposome fusion,lipofectin®, electroporation, viral infection, etc. The vectors maystably integrate into the genome of the host cell (for example, withretroviral introduction for mammalian cells, outlined herein), or mayexist either transiently or stably in the cytoplasm (i.e. through theuse of traditional plasmids, utilizing standard regulatory sequences,selection markers, etc.).

[0139] The vectors can be introduced simultaneously, or sequentially inany order. In a preferred embodiment, host cells containing the reporterconstruct are generated first, and preferably the reporter vector isintegrated into the genome of the host cell, for example, using aretroviral reporter vector. Once the components of the system are in thehost cell, the cell is subjected to conditions under which theselectable markers and fusion proteins are expressed. If a test proteinhas sufficient affinity to the switch ε region to activatetranscription, the detectable protein is produced, and cells containingthese proteins will survive drug selection and can be detected asoutlined above. The detectable protein will be produced at a measurablyhigher level than in the absence of a protein-nucleic acid interaction.Thus the determination of a protein-nucleic acid interaction isgenerally done on the basis of the presence or absence of the detectablegene(s).

[0140] In a preferred embodiment, once a cell with an altered phenotypeis detected, the cell is isolated from the plurality which do not havealtered phenotypes. This may be done in any number of ways, as is knownin the art, and will in some instances depend on the assay or screen.Suitable isolation techniques include, but are not limited to, drugselection, FACS, lysis selection using complement, cell cloning,scanning by Fluorimager, expression of a “survival” protein, inducedexpression of a cell surface protein or other molecule that can berendered fluorescent or taggable for physical isolation; expression ofan enzyme that changes a non-fluorescent molecule to a fluoroscent one;overgrowth against a background of no or slow growth; death of cells andisolation of DNA or other cell vitality indicator dyes; changes influorescent characteristics, etc. The preferred isolation techniques aredrug selection and FACS based on the expression of the detectable gene,with a preferred embodiment utilizing both simultaneously.

[0141] Once a cell with a protein-nucleic acid interaction is detectedand isolated, it is generally desirable to identify the test protein. Ina preferred embodiment, the test protein nucleic acid and/or the testprotein is isolated from the positive cell. This may be done in a numberof ways. In a preferred embodiment, primers complementary to DNA regionscommon to the vector, or to specific components of the library such as arescue sequence, are used to “rescue” the unique test sequence.Alternatively, the test protein is isolated using a rescue sequence.Thus, for example, rescue sequences comprising epitope tags orpurification sequences may be used to pull out the test protein, usingimmunoprecipitation or affinity columns. Alternatively, the test proteinmay be detected using mass spectroscopy.

[0142] Once a bioactive agent is identified, a number of things may bedone. In a preferred embodiment, the chacterization of the bioactiveagent is done. This will proceed as will be appreciated by those in theart, and generally includes an analysis of the structure, identity,binding affinity and function of the agent. Depending on the type ofagent, this may proceed in a number of ways. In a preferred embodiment,for example when the candidate agents have been introducedintracellularly using nucleic acid constructs, the candidate nucleicacid and/or the bioactive agent is isolated from the cells. This may bedone in a number of ways. In a preferred embodiment, primerscomplementary to DNA regions common to the retroviral constructs, or tospecific components of the library such as a rescue sequence, definedabove, are used to “rescue” the unique random sequence. Alternatively,the bioactive agent is isolated using a rescue sequence. Thus, forexample, rescue sequences comprising epitope tags or purificationsequences may be used to pull out the bioactive agent, usingimmunoprecipitation or affinity columns. Alternatively, the peptide maybe detected using mass spectroscopy.

[0143] Once rescued, the sequence of the bioactive agent and/orbioactive nucleic acid is determined. Similarly, candidate agents fromother chemical classes can be identified and characterized, for examplethrough the use of mass spectroscopy. This information can then be usedin a number of ways.

[0144] In a preferred embodiment, the bioactive agent is resynthesizedand reintroduced into the target cells, to verify the effect. This maybe done using retroviruses, or alternatively using fusions to the HIV-1Tat protein, and analogs and related proteins, which allows very highuptake into target cells. See for example, Fawell et al., PNAS USA91:664 (1994); Frankel et al., Cell 55:1189 (1988); Savion et al., J.Biol. Chem. 256:1149 (1981); Derossi et al., J. Biol. Chem. 269:10444(1994); and Baldin et al., EMBO J. 9:1511 (1990), all of which areincorporated by reference. Other techniques known in the art may be usedas well.

[0145] In a preferred embodiment, the sequence of a bioactive agent isused to generate more candidate bioactive agents. For example, thesequence of the bioactive agent may be the basis of a second round of(biased) randomization, to develop bioactive agents with increased oraltered activities. Alternatively, the second round of randomization maychange the affinity of the bioactive agent. Furthermore, it may bedesirable to put the identified random region of the bioactive agentinto other presentation structures, or to alter the sequence of theconstant region of the presentation structure, to alter theconformation/shape of the bioactive agent. It may also be desirable to“walk” around a potential binding site, in a manner similar to themutagenesis of a binding pocket, by keeping one end of the ligand regionconstant and randomizing the other end to shift the binding of thepeptide around.

[0146] Once identified and the biological activity is confirmed, thebioactive agent may be formulated. The compounds having the desiredpharmacological activity may be administered in a physiologicallyacceptable carrier to a host, as previously described. The agents may beadministered in a variety of ways, orally, parenterally e.g.,subcutaneously, intraperitoneally, intravascularly, etc. Depending uponthe manner of introduction, the compounds may be formulated in a varietyof ways. The concentration of therapeutically active compound in theformulation may vary from about 0.1-100 wt. %.

[0147] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

[0148] The following examples serve to more fully describe the manner ofusing the above-described invention, as well as to set forth the bestmodes contemplated for carrying out various aspects of the invention. Itis understood that these examples in no way serve to limit the truescope of this invention, but rather are presented for illustrativepurposes. All references cited herein are incorporated by reference intheir entirety.

EXAMPLES Example 1 Construction of ε Germline GFP/BFP Knock-in CellLines

[0149] Three different IgM⁺, EBV⁻ human B cells lines (CA-46, MC116,DND39, FIG. 4) that produce ε germline transcripts in the presence ofIL-4 will be transfected with a germline ε GFP or BFP knock-in construct(FIGS. 5B and 5C) and induced with IL-4. The cells will then be sortedby FACS for the appropriate reporter expression, GFP or BFP. Background(i.e. random integration) should be low since the construct mustintegrate downstream of an IL-4 inducible region in order to beactivated. Homologous recombination of the reporter construct will beconfirmed in fluorescent clones by genomic PCR using primers locatedwithin and immediately flanking the construct. For double knockouts,both GFP and BFP constructs will be transfected and cells sorted forexpression of both reporters.

[0150] It is possible that activation with IL-4 to identify homologousrecombined clones will result in events that move beyond the first phaseof ε switching, thus making the clones unusable for a screen identifyingblockers of this first step. For this case, we have designed a moretraditional construct containing an SV40 promoter-driven neomycinresistance gene which is flanked by loxP sites and inserted in theintron between the first and second ε constant coding exons (FIG. 5D).In addition, attached at the 3′ end of the long arm is a BFP reportergene driven by a constitutive promoter. B cell clones transfected withthis construct will be selected for integration by culturing them in thepresence of G418. The surviving cells lacking BFP will be sorted by FACS(the BFP at the 3′ end will be preferentially deleted during thehomologous recombination event). The remaining clones will be assessedfor homologous recombination by PCR. Clones containing homologousrecombined constructs will be exposed to the cre recombinase protein tomediate excision of the SV40 promoter/neomycin resistance gene in orderto eliminate promoter interference and potential ε promoter shutdown.Excision of the SV40 promoter/neomycin resistance gene fragment will beverified by subdividing clones into parent and daughter pools andre-selecting the latter pool in G418. The parental cells correspondingto G418 sensitive daughter cells will be subdivided again and tested forIL-4 inducible GFP expression. Parental stocks of the most inducibleclones will be used for subsequent peptide screening. Production of theknock-in cell line using this approach would provide a continuous sourceof IL-4 inducible cells and would circumvent any down-regulationassociated with IL-4 pre-treatment.

Example 2 Creation and Screening of Candidate Bioactive Agents inKnock-in Cell Lines

[0151] A candidate bioactive agent library, in this case a peptidelibrary, will be packaged into infectious viral particles as outlinedbelow. A preferred library is a mixture of random peptide sequences withand without a nuclear localization sequence (NLS) upstream of a reportergene to identify infected cells and relative peptide expression (seeFIG. 6).

[0152] Each screen will start with production of the primary retroviruspeptide library, as is generally shown in FIG. 7. This is generally doneas outlined in PCT US97/01019 and PCT US97/01048, both of which areexpressly incorporated by reference. In general, this is done asfollows. On day 1, the Phoenix cells are seeded in 10 cm plates at 5×106cells in 6 ml (DMEM+10% FBS+Pen/Strep) per plate the day beforetransfection. Day 2: allow all reagents to reach room temperature 30min. before starting. Add 50 mM chloroquine at 8 μl/plate (50 μM final)before preparing the transfection solution. Mix CaPO₄ reagents in 15 mlpolypropylene tube: per plate: 10 μg DNA, 122 μl 2M CaCl₂, 876 μl H₂O,1.0 ml 2×HBS. Add 2×HBS and depress the expulsion button completely tobubble air through the mix for 10 secs. Immediately add mixture gentlydropwise to plate. Incubate 3-8 hours. Remove medium and replace with6.0 ml DMEM-medium. Day 3: Change medium again to 6.0 mls of mediumoptimal for the cells to be infected. Move to 32° C. either in themorning or afternoon depending on the Phoenix cell confluency andwhether you will infect at 48 or 72 hrs after transfection. Day 4 or 5:Collect virus supernatant from transfected plates (6.0 ml) into 50 mltubes and add protamine sulfate to a final concentration of 5 μg/ml.Pass through a 0.45 μm filter. Count target cells and distribute 10⁷cells per 10 cm plate transfected to 50 ml tubes and pellet 5 min.Resuspend each pellet of target cells in virus supernatant and transferto a 6 well plate at 1.0-1.2 ml per well. Seal plate with parafilm andcentrifuge at RT for 30-90 min. at 2500 RPM. Remove parafilm andincubate plate over night at 37° C. Day 5: Collect and pellet each wellof target cells. Resuspend in 3 ml medium and transfer back to the same6well plate. Infection can be repeated by refeeding the Phoenix cellswith 6 ml fresh medium and reinfecting the same cells again up to 3times to increase % of cells infected (for instance at 48, 56, and 72hours). Day 7 or Day 8: At 48 to 72 hrs. post infection, target cellsare ready to analyze for expression.

[0153] This primary library will be used to infect at least 10⁹ knock-incells. After infection, the cells will be stimulated with IL-4 and twodays later, peptide-containing cells (identified by the fluorescentreporter) that are negative for the knock-in reporter (ie. where thereis ε promoter inhibition) will be sorted by FACS. This enriched,knock-in reporter negative population will be subjected to RT-PCR toamplify the integrated peptide sequences. The PCR material will be usedto construct a new “enriched” retrovirus peptide library to initiate thenext screening round.

[0154] It will take approximately 5-7 rounds of enrichment to identifyindividual sequences capable of inhibiting the germline ε promoter, asoutlined below using an iterative screening equation. R = ρ + ( Q + ∑ i= 0 ∞     β  ( 1 + ) ρ ) + v

[0155] The above equation mathematically models screening efficiency andprovides a guideline for monitoring enrichment for inhibitory peptides.R=ratio of true positive cells over the total number of cells screenedper round of selection; ν=frequency of true positive cells (i.e. # ofcells expressing peptide inhibitors of IgE switch/synthesis);ε=frequency of non-heritable false-positive cells (i.e. # of cells inwhich IgE switch/synthesis is inhibited due to stimulation/screeninginefficiencies, but are IgE positive in subsequent selection rounds);ρ=number of rounds of selection/enrichment applied to library screen;Q=initial frequency of cells with an heritable false-positive phenotype(i.e. dominant-negative somatic mutation in cells that prevent IgEswitch/synthesis); β=frequency of false-positives incurred by or duringthe selection/enrichment process.

[0156] Since we amplify enriched peptides by RT-PCR after each selectionround, the equation can be simplified to R = v ρ + Q + v

[0157] By plugging in empirically-derived or estimated values for thevariables, an estimate of how many selection rounds must be applied to alibrary before enrichment for IgE inhibitory peptide becomes apparent.

[0158] For the purposes of our screens, we engineer and select reportercell lines in which the values of and Q are low to minimize the numberof screening rounds necessary to observe rare positive peptide “hits”.

[0159] For example, IL-4 treatment upregulates the IgE switch reporterin 97% of cells, therefore ε=0.03. Of the uninduced cells, a secondround of stimulation indicates that less than 0.01% of the startingpopulation contain heritable false positives, therefore Q<0.0001. Aconservative estimate of IgE inhibitory peptides in the startingpopulation is 1/10⁸, therefore v−10⁻⁸. Solving the equation for thenumber of selection rounds required to enrich to 50% true positive hits. . .$0.5 = {\left. \frac{10^{- 8}}{(0.03)^{\rho} + 10^{- 3} + 10^{- 8}}\rightarrow\rho \right. = {5\quad {rounds}}}$

[0160] The most important factor that influences the number ofenrichment rounds necessary to identify individual peptide hits is theratio between the real positive peptide hits in the original library andthe heritable false positives. The frequency of real positive peptidehits is dependent upon the qualitative ability of the peptide to accessand, in the correct conformation, bind to regulatory domains on proteinsin the pathway of interest. Thus, preferably, multiple scaffoldingstructures are used for presentation of random peptide surfaces and alsodifferent localization sequences fused to those peptide structures.Enrichment of real positive peptides becomes less efficient with falsepositive rates above 2%. For this reason, great emphasis is placed ondeveloping robust reporter constructs and cell lines.

[0161] Uneven RT-PCR amplification may decrease overall amplification ofreal peptides hits from one round to another. This is overcome byadditional rounds of library enrichment and is why RT-PCR amplificationis carefully monitored after each round of screening.

Example 3 Screening for Inhibitors of IgE Secretion in Cells that haveAlready Switched

[0162] After B cells have switched to production of IgE, there areseveral factors that determine when they will secrete IgE. By screeningfor peptide inhibitors of surface IgE expression, proteins that regulateIgE transcription, translation, assembly and trafficking may beidentified.

[0163] The IgE⁺ cell line, U266, expresses IgE on the surface and alsosecretes IgE. Antibodies against surface IgE heavy and light chains havebeen obtained and both are used to fluorescently mark IgE positivecells. The U266 line is consistently greater than 98.5% positive formembrane IgE.

[0164] Peptide library screening and target identification: The peptidelibrary and enrichment protocols identical to those described in Example2. As well, peptide hit validation and corresponding target proteinidentification will be performed as described in Example 2.

[0165] Development of an ε-heavy chain GFP/BFP knock-in cell linederivative of U266: The cytoplasmic tail of the ε-heavy chain in U266cells will be engineered by homologous recombination to encode a GFP/BFPreporter as shown in FIG. 8. This will produce a cell line that isfluorescent when ε-heavy chains are produced. The GFP will be attachedto the secretory exon to label secretory IgE heavy chains green. The BFPwill be attached to the M2 exon to label membrane IgE heavy chains blue.This will allow discrimination of mRNA processing and translationbetween secretory versus membrane ε-heavy chain transcripts.

[0166] The construct will contain an SV40 promoter-driven neomycinresistance gene which is flanked by loxP sites and inserted in theintron between the CH3 and CH4 exons (FIG. 8). In addition, the HSV-TKgene will be cloned 3′ of the longer homologous sequence region. U266cells transfected with this construct will be selected for integrationby culturing them in the presence of G418. The surviving cells will becultured in ganciclovir to select against cells containing the HSV-TKgene (the HSV-TK gene at the 3′ end will be deleted during the desiredhomologous recombination event). The remaining clones will be assessedfor homologous recombination by PCR. Clones containinghomologously-recombined constructs will be transfected with cre tomediate excision of the SV40 promoter/neomycin resistance gene in orderto eliminate promoter interference. Excision will be verified bysubdividing clones into parent and daughter pools and re-selecting thelatter pool in G418. The parental cells corresponding to G418 sensitivedaughter cells will be subdivided again and tested for GFP and BFPexpression. Parental stocks of the most inducible clones will be usedfor of subsequent screening.

Example 4 Development of an ε Promoter GFP Reporter Cell Line

[0167] The induction of the ε promoter in response to IL-4/13 is thefirst recognizable step necessary for the switch to IgE. Blockingactivation of this promoter should prevent B cells from switching toIgE. Inhibitors are predicted to interfere with IL-4/13 signaling aswell as nuclear transcription of the ε germline gene.

[0168] Three IgM⁺, EBV⁻ human B cells lines (CA-46, MC116, and DND39;see FIG. 4) that produce ε germline transcripts in the presence of IL-4will be infected with the following construct: a retroviral vectorcontaining an IL-4 responsive 600 bp fragment of the ε promoter in thereverse orientation followed by a splice site, GFP encoding sequence anda poly-adenylation sequence (FIG. 10). Briefly, cells will be infectedwith the reporter construct and induced with IL-4. The cells will thenbe sorted by FACS for GFP reporter expression. The IL-4 will be removedand the cells will be sorted for the absence of reporter fluorescence.From these sorts, several clones will be established that turn on thereporter in the presence of IL-4, indicating activation of the germlineε promoter.

Example 5 Screening of Candidate Agents using Reporter Cell Line

[0169] The cell line of Example 4 is infected infected with a peptidelibrary as described above. The peptide library is packaged intoinfectious viral particles (see FIG. 7). The library is a mixture ofrandom peptide sequences with and without a nuclear localizationsequence (NLS) upstream of a reporter gene to identify infected cellsand relative peptide expression (FIG. 6).

[0170] Each screen will start with production of the primary retroviruspeptide library. This primary library will be used to infect at least10⁹ ε promoter reporter cells. After infection, the cells will bestimulated with IL-4 and two days later, the FACS will sortpeptide-containing, reporter negative cells (i.e. where there is εpromoter inhibition). This enriched, reporter negative population willbe subjected to RT-PCR to amplify the integrated peptide sequences. ThePCR material will be used to construct a new “enriched” retroviruspeptide library to initiate the next screening round.

[0171] It will take approximately 5-7 rounds of enrichment to identifyindividual sequences capable of inhibiting the germline ε promoter (seediscussion above regarding the statistics associated with enrichment).The most important factor that influences the number of enrichmentrounds necessary to identify individual peptide hits is the ratiobetween real positive peptide hits in the original library and heritablefalse positives. The frequency of real positive peptide hits isdependent upon the qualitative ability of the peptide to get to and, inthe correct conformation, bind to the regulatory domains on proteins inthe pathway of interest. This is why we use multiple scaffoldingstructures for presentation of random peptide surfaces and alsodifferent localization sequences fused to those peptide structures(Appendix B). Enrichment of real positive peptides becomes lessefficient with false positive rates above 2%. For this reason, greateffort is placed in developing robust reporter constructs and celllines.

[0172] Once enrichment is achieved and individual peptide sequences areshown to effect inhibition of ε promoter activation in an independentassay, they will be introduced into a standard set of secondary andorthogonal assays. Many of these assays will be performed in primary Bcells to test the specificity and physiologic characteristics of thepeptide inhibitor.

Example 6 Generation of an ε Promoter Survival Cell Line

[0173] Three different IgM⁺, EBV⁻ human B cells lines that produce εgermline transcripts in the presence of IL-4 will be infected with asurvival construct carrying a death gene and a drug selectable marker(FIG. 10). Briefly, the retroviral construct consists of the 600 bp IL-4inducible ε promoter downstream of a self-inactivating (SIN) LTR,followed by a chimeric FAS receptor (FASr), the self-cleaving peptide 2aand, lastly, the drug-selectable puromycin resistance gene. The chimericreceptor is composed of the mouse FASr external domain and the humanFASr transmembrane and cytoplasmic domains. A mouse specific anti-FASrantibody can be used which will bind only activated FASr produced by thesurvival construct. The 2a self-cleaving peptide allows equimolaramounts of the chimeric FASr and puromycin to be produced in the cell.

[0174] IgM⁺ B cell lines infected with this construct in the presence ofIL-4 will produce CD95, as well as puromycin resistance. Upon drugselection with puromycin, only cells containing IL-4 activated εpromoters will survive. The remaining cells are infected with thepeptide libraries and, when cultured in the presence of IL-4 andanti-FAS ((αCD95) monoclonal antibodies, will express the chimeric FASreceptor and apoptose unless their ε promoter has been blocked by alibrary peptide.

[0175] If problems arise due to over-expression of the chimeric FASrresulting in self-activation, other external domains will be used. Wehave already engineered a chimeric FASr containing the murine CD8external domain as an alternative (FIG. 10). If overexpression of thechimeric FASr results in self-activation, we have designed analternative strategy in which the proposed construct contains the GFPgene in lieu of the puromycin resistance gene (FIG. 10). Due to the mildtranscriptional leakiness inherent to all SIN retroviral vectors, asmall percentage of IgM+ B cell clones infected with this construct willexpress low, detectable levels of GFP. These cells can be single-cellcloned by FACS, split into parent and daughter pools and tested for IL-4inducible FASr expression-dependent apoptosis. Parent stocks of the mostefficiently killed daughter cells will provide a continuous cell sourcefor subsequent peptide screening assays. In addition, FASr ligation canbe used to potentiate cell death and thus diminish background cellsurvival.

[0176] Additionally, IL-4 stimulation has been reported to diminishFAS-induced apoptosis in certain B-cell lines. To circumvent thispotential difficulty, common suicide genes including Herpes SimplexVirus Thymidine Kinase (HSV-TK) or human cytochrome P450 2B1 inconjunction with ganciclovir or cyclophosphamide treatment,respectively, can replace FASr-mediated death (FIG. 10). Alternatively,cell cycle arrest genes such as p21 can be used in place of toxic geneproducts (FIG. 10). In this way, cells expressing peptides which preventIL-4 induced overexpression of p21 will have a selective growthadvantage and will quickly dominate the culture.

Example 7 Screening in ε Promoter Survival Cells

[0177] Using a peptide library generated as outlined above, the IgM⁺ Bcell lines described in Example 6 are infected with the survivalconstruct. Leaky cells (constitutive expression of the ε promoter) willbe removed by incubation with the anti-mouse FASr antibody. Next, thecells are incubated in the presence of the inducer, IL-4, and the drugselection compound, puromycin. Cells that contain a construct that isinducible by IL-4 will be resistant and survive. This produces apopulation with an exogenous ε promoter that is IL-4 inducible. Thepeptide library is introduced into these cells and two days later theyare induced with IL-4 in the presence of anti-mouse FASr monoclonalantibody. Cells carrying peptides that inhibit induction of theengineered ε promoter fragment will not produce the chimeric FASr andwill survive. After the survivors grow out (approximately 1 week), theywill again be subjected to IL-4 and the anti-FASr treatment. The genesencoding the peptides responsible for the survivors will be rescued byRT-PCR and used to generate an enriched retroviral library. Theidentification of individual inhibitory peptides should occur in only3-4 rounds since the false positive background for survival screens islower than for FACS-based screening. Once enrichment is achieved andindividual peptide sequences are independently shown to inhibit εpromoter activation, these sequences will be introduced into a standardset of secondary and orthogonal assays.

Example 8 One-hybrid Screens for Identification of Proteins that Bind toSwitch ε Region

[0178] Recombinase proteins that bind to the Sε region mediate the DNArearrangement that generates a functional ε heavy chain. They may bespecific for ε switching cells or may bind to other proteins that targetthem specifically to the Sε region. Breakpoints in the recombination ofthe switch ε region to the switch μ region occur in a limited area ofthe switch ε region. Two stretches of the switch ε region spanning themajority of breakpoints will be used as bait in a one-hybrid screen(FIG. 2b). The cDNA libraries to be used are derived from the IgEpositive cell line U266 (the assumption here is that the U266 line stillcontains the switch recombinase; certainly, the recombinase is turnedoff in plasma cells) and from human peripheral blood lymphocytesstimulated in vitro to switch with a high frequency to IgE.

[0179] The screening is summarized in FIG. 3. The methods are asfollows: Two stretches of the switch ε region were cloned (FIG. 2A) intoEcoR I/Xba I sites of pHISi-1 (Clontech) to construct a HIS reportervector pIgE-HIS. In this construct, HIS expression is under the controlof a minimal promoter and proteins binding to the switch ε region.Similarly, a second LacZ reporter is constructed by inserting twostretches of switch ε region into the EcoR I/Xho I sites of pLacZi toconstruct pIgE-LacZ.

[0180] The pIgE-HIS was linearized at an Afl II site and integrated intoyeast strain YM4271 (MATa, ura3-52, his3-200, ade2-101, lys2-801,leu2-3, 112, trpl-901, tyrl-501, gal4-Δ512, gal80-Δ538, ade5::hisG) toconstruct the first yeast reporter strain YIgE-HIS. SD-H plates wereused to select for integrated reporters. The yeast strain YIgE-HlS wastested on SD-H+3AT plates to determine the optimal concentration of 3ATto suppress basal level HIS expression from the minimal promoter.

[0181] The pIgE-LacZ plasmid was linearized at an Nco I site andintegrated into the yeast strain YIgE-HIS to construct a dual reporterstrain YIgE-HL. SD-U plates were used to select for cells with dualreporters integrated. The dual reporter strain will be used fortransformation by the U266 cDNA library (it is assumed that the U266line still contains the switch recombinase) and the IgE switching PBLcDNA library. At least 20 million transformants from each library willbe screened on SD-LH+3AT plates. Clones that can grow up and turn blueon SD-LH+3AT plates will be grown up in SD-L liquid medium for plasmidretrieval. Retrieved cDNA clones will be further tested using in vitrobinding assays.

We claim:
 1. A method of screening for bioactive agents capable ofinhibiting an IL-4 inducible ε promoter, said method comprising a)combining a candidate bioactive agent and a cell comprising a fusionnucleic acid comprising: i) an IL-4 inducible ε promoter; and ii) areporter gene; b) inducing said promoter with IL-4; and c) detecting thepresence or absence of said reporter protein; wherein the absence ofsaid reporter protein indicates that said agent inhibits said IL-4inducible ε promoter.
 2. A method according to claim 1, wherein saidreporter gene encodes a fluorescent protein.
 3. A method according toclaim 1, wherein said reporter gene encodes a death protein that isactivated by the introduction of a ligand, and said method furthercomprises adding said ligand to said cell.
 4. A method according toclaim 3, wherein said fusion nucleic acid further comprises a differentreporter gene.
 5. A method according to claim 3 wherein said deathprotein is a Fas receptor and said ligand is Fas.
 6. A method accordingto claim 5 wherein said Fas receptor is a chimeric receptor comprising:a) the extracellular domain of murine Fas receptor; and b) the cytosolicdomain of human Fas receptor.
 7. A method according to claim 3 whereinsaid death protein is a chimeric protein comprising: a) theextracellular domain of a ligand-activated dimerizing receptor; and b)the cytosolic domain of a Fas receptor.
 8. A method according to claim 7wherein said ligand-activated dimerizing receptor is selected from thegroup consisting of CD8 receptor, erythropoeitin receptor,thrombopoeitin receptor, growth hormone receptor, Fas receptor, plateletderived growth hormone receptor, epidermal growth factor receptor,leptin receptor, an interleukin receptor, low-density lipoproteinreceptor, prolactin receptor, and transferrin receptor.
 9. A methodaccording to claim 1 wherein said fusion nucleic acid comprises anexogeneous IL-4 inducible ε promoter.
 10. A method according to claim 1wherein said fusion nucleic acid comprises an endogeneous IL-4 inducibleε promoter.
 11. A method according to claim 1 wherein said combining isdone by introducing a retroviral vector comprising nucleic acid encodingsaid candidate bioactive agent to said cell.
 12. A method according toclaim 11 wherein a library of retroviral vectors comprising a library ofcandidate bioactive agents is added to a population of cells.
 13. Amethod according to claim 11 wherein said retroviral vector furthercomprises nucleic acid encoding a fluorescent label.
 14. A methodaccording to claim 2 or 13 wherein said detecting is done using a FACSmachine.
 15. A cell line for screening selected from the groupconsisting of CA-46 and MC-116, said cell line comprising a fusionnucleic acid comprising: a) an IL-4 inducible ε promoter; and b) areporter gene.
 16. A method of screening for bioactive agents capable ofmodulating IgE production, said method comprising: a) combining acandidate bioactive agent and a cell capable of expressing IgE; b)determining the amount of IgE produced in said cell; wherein a change inthe amount of IgE as compared to the amount produced in the absence ofsaid candidate agent indicates that said agent modulates IgE production.17. A method according to claim 16 wherein said modulation is a decreasein the amount of IgE.
 18. A method according to claim 16 wherein saidcell comprises a IgE fusion protein comprising: a) the ε heavy chain;and b) a fluorescent protein.
 19. A method according to claim 16 whereinsaid combining is done by introducing a retroviral vector comprisingnucleic acid encoding said candidate bioactive agent to said cell.
 20. Amethod according to claim 19 wherein a library of retroviral vectorscomprising a library of candidate bioactive agents is added to apopulation of cells.
 21. A method according to claim 19 wherein saidretroviral vector further comprises nucleic acid encoding a fluorescentlabel.
 22. A method according to claim 16 wherein said detecting is doneby the addition of a fluorescent antibody against IgE.
 23. A method ofscreening for bioactive agents capable of inhibiting a promoter ofinterest, said method comprising a) combining a candidate bioactiveagent and a cell comprising a fusion nucleic acid comprising: i) apromoter of interest; and ii) a reporter gene comprising a death genethat is activated by the introduction of a ligand; b) optionallyinducing said promoter; c) introducing said ligand to said cell; and d)detecting the presence of said cell, wherein the presence of said cellindicates that said agent inhibits said promoter.
 24. A method accordingto claim 23 wherein said fusion nucleic acid further comprises adifferent reporter gene.
 25. A method according to claim 23 wherein saidpromoter is an endogeneous promoter.
 26. A method according to claim 23wherein said promoter is an exogeneous promoter.
 27. A compositioncomprising: a) a test vector comprising: i) a first selection gene; ii)a fusion gene comprising: 1) a first sequence encoding a transcriptionalactivation domain; and 2) a second sequence encoding a test protein; andb) a reporter vector comprising: i) a first detectable gene; ii) all orpart of the switch ε sequence, which upon binding of saidtranscriptional activation domain due to a protein-nucleic acidinteraction between said test protein and said switch ε sequence, willactivate transcription of said first detectable gene.
 28. A method ofidentifying proteins that bind to all or part of the switch ε region ofFIG. 2B, said method comprising: a) providing a host cell comprising thecomposition of claim 27; b) subjecting said host cell to conditionsunder which the fusion gene is expressed to produce a fusion protein;and c) determining whether a protein-nucleic acid interaction betweensaid fusion protein and said switch ε sequence occurred.